Pharmaceutical compositions and their use for treatment of cancer and autoimmune diseases

ABSTRACT

Described herein are combination therapies for cancer (such as lymphoid malignancies) and immune diseases (such as autoimmune diseases and inflammatory diseases). The therapies comprise the combined use of inhibitors of BTK, mTOR kinase, and Bcl-2 or their signaling pathways, and immunomodulatory drugs. Also described are pharmaceutical compositions and kits comprising these inhibitors.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of International ApplicationPCT/US2017/037783, filed Jun. 15, 2017, which is a continuation-in-partof U.S. patent application Ser. No. 15/183,340, filed Jun. 15, 2016, nowU.S. Pat. No. 9,717,745, which is a continuation-in-part ofInternational Application PCT/CN2016/000149, filed Mar. 18, 2016, whichclaims priority from Chinese Patent Application No. 201510119944.5,filed Mar. 19, 2015. This application is also a continuation-in-part ofU.S. application Ser. No. 15/628,143, filed Jun. 20, 2017, now U.S. Pat.No. 10,098,900, which is a division of U.S. application Ser. No.15/183,340, filed Jun. 15, 2016, now U.S. Pat. No. 9,717,745, which is acontinuation-in-part of International Application PCT/CN2016/000149,filed Mar. 18, 2016, which claims priority from Chinese PatentApplication No. 201510119944.5, filed Mar. 19, 2015. The contents of allof the aforementioned priority applications are incorporated herein byreference in their entirety.

BACKGROUND OF THE INVENTION

Cancer treatment has evolved over time to become more targeted and lesstoxic to the patient. Traditional chemotherapy often has a high level ofsystemic toxicity. Targeted therapy uses small molecules or biologics(e.g., therapeutic antibodies) to inhibit the activity of a selectedcellular protein involved in cancer development, and causes much lessside effect than traditional chemotherapy. Immunotherapies such as thosetargeting immune checkpoints (e.g., PD-1 and PD-L1) and those involvingchimeric antigen receptor T (CAR-T) cells aim to bolster the patient'sown anti-cancer immune defense, and have emerged as a promising newtreatment paradigm.

One of the cellular proteins that have been targeted in cancer therapyis Bruton tyrosine kinase (BTK). BTK is a member of the Tec family ofprotein tyrosine kinases. BTK has domains with pleckstrin homology (PH),Tec homology (TH), Src homology 3 (SH3), Src homology 2 (SH2), andtyrosine kinase or Src homology 1(TK or SH1) (Akinleye et al.,“Ibrutinib and novel BTK inhibitors in clinical development,” Journal ofHematology & Oncology, 2013, 6:59). Proper expression of the BTK gene indifferent lymphoid regions plays a key role in normal B-celldevelopment. BTK is also involved in signal transduction pathways for Bcell activation and survival (Kurosaki, “Molecular mechanisms in B cellantigen receptor signaling,” Curr OP Imm, 1997, 9(3):309-18).

BTK functions downstream of multiple receptors, including B-CellReceptor (BCR), receptors for growth factors and chemokines, and innateimmune receptors. BTK initiates a broad range of cellular processes,such as cell proliferation, survival, differentiation, motility,adhesion, angiogenesis, cytokine production, and antigen presentation,and plays an important role in hematological malignancies and immunedisorders. In a mouse model for chronic lymphocytic leukemia (CLL), BTKexpression levels were shown to set the threshold for malignanttransformation; BTK overexpression accelerated leukemia and increasedmortality (Kil et al., “Bruton's tyrosine kinase mediated signalingenhances leukemogenesis in a mouse model for chronic lymphocyticleukemia,” Am J Blood Res, 2013, 3(1):71-83).

Ibrutinib (also known commercially as IMBRUVICA®) was the first BTKinhibitor approved by the United States Food and Drug Administration fortreating mantle cell lymphoma (MCL), chronic lymphocytic leukemia (CLL),and Waldenstrom's macroglobulinemia (WM). In general, however, theselectivity of known BTK inhibitors is not ideal—they inhibit not onlyBTK, but also various other kinases (such as ETK, EGF, BLK, FGR, HCK,YES, BRK and JAK3, etc.). Known BTK inhibitors also produce a variety ofderivatives. These characteristics of known BTK inhibitors lead to adecrease in therapeutic efficacy and an increase in side effects. Thepharmacokinetics of known BTK inhibitors also needs to be improved.Indeed, significant variations in bioavailability of ibrutinib have beenobserved clinically among patients (Marostica et al., “Populationpharmacokinetic model of ibrutinib, a Bruton tyrosine kinase inhibitor,in patients with B cell malignancies,” Cancer Chemother Pharmacol, 2015,75:111-121).

SUMMARY OF THE INVENTION

The invention relates to methods of inhibiting cancer cells and treatingcancer, and to methods of inhibiting lymphocytes (e.g., B cells) andtreating immune disorders. In these methods, a BTK inhibitor such as amulti-fluoro-substituted pyrazolopyrimidine compound described hereinand an inhibitor of mammalian target of rapamycin (mTOR) are used. Incertain embodiments, a third drug, such as an inhibitor of B-celllymphoma 2 (Bcl-2) or PI3 kinase, or an immunomodulatory drug (IMiD), isalso used. Applicant has discovered that the particular combinations ofdrugs described herein are unexpectedly high synergistic effects and caneffectively overcome drug resistance and disease recurrence. Combinationtherapies described herein are much safer than monotherapy due to thelower doses used and can shorten treatment cycle because of bettertherapeutic effects.

One aspect of the invention described herein relates to a method fortreating a cancer, such as a lymphoid malignancy (e.g., chroniclymphocytic leukemia, Waldenstrom Macroglobulinemia, mantle celllymphoma), comprising administering to a subject in need thereof atherapeutically effective amount of (i) a BTK inhibitor, (ii) an mTORkinase inhibitor, and (iii) a Bcl-2 inhibitor or an IMiD. In someembodiments, the lymphoid malignancy is multiple myoloma, which iscurrently treated with IMiD or its existing combinations with otherdrugs, but there is still significant unmet medical needs for thisdisease.

Another aspect of the invention relates to method for treating an immunedisorder, such as an autoimmune disease (e.g., rheumatoid arthritis andsystemic lupus erythematosus), comprising administering to a subject inneed thereof a therapeutically effective amount of (a) a Bruton tyrosinekinase (BTK) inhibitor, and (b) a mammalian target of rapamycin (mTOR)kinase inhibitor.

In some embodiments, the BTK inhibitor is selected from the groupconsisting of a compound represented by Formula I, II, Ia, Ib, IIa, orIIb, ibrutinib, acalabrutinib, BGB-3111, spebrutinib, ONO-4059, HM71224,RN486, CNX-774, CGI-11746, and other BTK inhibitors, and enantiomers,diastereomers, and pharmaceutically acceptable salts thereof, whereinthe aforementioned Formulae are:

wherein:

each R¹ is F;

R² is F;

R³ is H or D;

n is 1, 2, 3 or 4; and

m is 1 or 2,

or an enantiomer or diastereomer thereof, or a pharmaceuticallyacceptable salt or prodrug thereof.

In some embodiments, the BTK inhibitor is selected from the groupconsisting of: Compound 1, Compound 2, Compound 3, Compound 4, Compound5, Compound 6, Compound 7, Compound 20, and enantiomers, diastereomers,and pharmaceutically acceptable salts thereof.

In some embodiments, the BTK inhibitor is Compound 3 or an enantiomer,diastereomer, or pharmaceutically acceptable salt thereof.

In some embodiments, the BTK inhibitor is Compound 5 or an enantiomer,diastereomer, or pharmaceutically acceptable salt thereof.

In some embodiments, the BTK inhibitor is ibrutinib or apharmaceutically acceptable salt thereof.

In some embodiments, the mTOR kinase inhibitor is selected from thegroup consisting of everolimus, rapamycin, temsirolimus, ridaforolimus,XL388, GDC-0349, AZD2014, AZD8055, GSK105965, MLN0128, PI-103,NVP-BEZ235, WJD008, XL765, SF-1126, Torin1, PP242, PP30, Ku-0063794,WYE-354, WYE-687, WAY-600, INK128, OSI-027, other known mTOR kinaseinhibitors, and pharmaceutically acceptable salts thereof.

In some embodiments, the mTOR kinase inhibitor is everolimus or apharmaceutically acceptable salt thereof.

In some embodiments, the mTOR kinase inhibitor is rapamycin or apharmaceutically acceptable salt thereof.

In some embodiments, the IMiD is lenalidomide, pomalidomide,thalidomide, CC-112, or CC-220, or a pharmaceutically acceptable saltthereof.

In some embodiments, the Bcl-2 inhibitor is selected from the groupconsisting of venetoclax (ABT-199), navitoclax, ABT-737, TW-37,sabutoclax, obatoclax, other known Bcl-2 inhibitors, andpharmaceutically acceptable salts thereof.

In some embodiments, the Bcl-2 inhibitor is venetoclax or apharmaceutically acceptable salt thereof.

In some embodiments, the method comprises administering to a cancerpatient a therapeutically effective amount of (a) ibrutinib or apharmaceutically acceptable salt thereof, (b) everolimus or apharmaceutically acceptable salt thereof, and (c) venetoclax or apharmaceutically acceptable salt thereof.

In some embodiments, the method comprises administering to a cancerpatient a therapeutically effective amount of (a) ibrutinib or apharmaceutically acceptable salt thereof, (b) rapamycin or apharmaceutically acceptable salt thereof, and (c) venetoclax or apharmaceutically acceptable salt thereof.

In some embodiments, the method comprises administering to a cancerpatient a therapeutically effective amount of (a) Compound 3 or anenantiomer, diastereomer, or pharmaceutically acceptable salt thereof,(b) everolimus or a pharmaceutically acceptable salt thereof, and (c)venetoclax or a pharmaceutically acceptable salt thereof.

In some embodiments, the method comprises administering to a cancerpatient a therapeutically effective amount of (a) Compound 3 or anenantiomer, diastereomer, or pharmaceutically acceptable salt thereof,(b) rapamycin or a pharmaceutically acceptable salt thereof, and (c)venetoclax or a pharmaceutically acceptable salt thereof.

In some embodiments, the method comprises administering to a cancerpatient a therapeutically effective amount of (a) Compound 5 or anenantiomer, diastereomer, or pharmaceutically acceptable salt thereof,(b) everolimus or a pharmaceutically acceptable salt thereof, and (c)venetoclax or a pharmaceutically acceptable salt thereof.

In some embodiments, the method comprises administering to a cancerpatient a therapeutically effective amount of (a) Compound 5 or anenantiomer, diastereomer, or pharmaceutically acceptable salt thereof,(b) rapamycin or a pharmaceutically acceptable salt thereof, and (c)venetoclax or a pharmaceutically acceptable salt thereof.

In some of the above embodiments, the Bcl-2 inhibitor such as venetoclaxmay be replaced with an IMiD such as pomalidomide, thalidomide, orlenalidomide.

In some embodiments, the method comprises administering to a patientwith an immune disorder a therapeutically effective amount of (a)ibrutinib, Compound 3, or Compound 5 and (b) everolimus or rapamycin.

In some embodiments, each of the BTK inhibitor, the mTOR kinaseinhibitor, and the Bcl-2 inhibitor or IMiD are administeredsequentially, in any order.

In some embodiments, each of the BTK inhibitor, the mTOR kinaseinhibitor, and the Bcl-2 inhibitor or IMiD are administered together,e.g., administered in three pharmaceutical compositions concurrently, oras in the same, co-formulated pharmaceutical composition.

In some embodiments, each of the BTK inhibitor, the mTOR kinaseinhibitor, and the Bcl-2 inhibitor or IMiD are orally administered tothe subject one or more times daily.

In some embodiments, the daily dose of the BTK inhibitor is between 5 mgand 1000 mg. In some embodiments, the daily dose of the mTOR kinaseinhibitor is between 0.1 mg and 10 mg. In some embodiments, the dailydose of the IMiD is between 1 mg and 30 mg. In some embodiments, thetotal daily dose of the BTK inhibitor, the mTOR kinase inhibitor, andthe IMiD is 300 mg or less.

In some embodiments, the daily dose of the BTK inhibitor is between 5 mgand 1000 mg. In some embodiments, the daily dose of the mTOR kinaseinhibitor is between 0.1 mg and 10 mg. In some embodiments, the dailydose of the Bcl-2 inhibitor is between 10 mg and 1000 mg. In someembodiments, the total daily dose of the BTK inhibitor, the mTOR kinaseinhibitor, and the Bcl-2 inhibitor is 500 mg or less.

In some embodiments, the cancer is a B-cell malignancy selected from thegroup consisting of small lymphocytic lymphoma (SLL), chroniclymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL),Waldenström Macroglobulinemia (WM), follicular lymphoma (FL), mantlecell lymphoma (MCL), marginal zone lymphoma (MZL), and multiple myeloma(MM).

In some embodiments, the cancer is selected from the group consisting ofbrain tumors, bladder cancer, stomach cancer, ovarian cancer, pancreaticcancer, breast cancer, head and neck cancer, cervical cancer,endometrial cancer, colorectal cancer, kidney cancer, esophageal cancer,adenocarcinoma, thyroid cancer, bone cancer, skin cancer, colon cancer,female reproductive tract tumors, lymphomas, and testicular cancer.

In some embodiments, the method is effective to reduce average tumorvolume of TMD-8 lymphoma xenograft in SCID mice by at least 80%, after14 days of treatment with at a total daily dose of the BTK inhibitor,the mTOR kinase inhibitor, and the Bcl-2 inhibitor of 20 mg/kg or less.

Another aspect of the invention described herein relates to apharmaceutical composition comprising a BTK inhibitor, an mTOR kinaseinhibitor, a Bcl-2 inhibitor, and a pharmaceutically acceptable carrier.

A further aspect of the invention described herein relates to apharmaceutical kit comprising a first oral dosage of a BTK inhibitor, asecond oral dosage of an mTOR kinase inhibitor, and a third oral dosageform of a Bcl-2 inhibitor or an IMiD. In some embodiments, thepharmaceutical kit further comprises instructions for administering saiddosage forms to treat cancer in a subject in need thereof.

A further aspect of the invention described herein relates to a methodfor treating a cancer or an autoimmune disease, comprising administeringto a subject in need thereof a therapeutically effective amount of (a) aBruton's tyrosine kinase (BTK) inhibitor and (b) a mammalian target ofrapamycin (mTOR) kinase inhibitor.

In some embodiments, the method comprises administering to the subject atherapeutically effective amount of (a) ibrutinib or a pharmaceuticallyacceptable salt thereof and (b) everolimus or a pharmaceuticallyacceptable salt thereof. In some embodiments, the method comprisesadministering to the subject a therapeutically effective amount of (a)Compound 3 or a pharmaceutically acceptable salt thereof and (b)everolimus or a pharmaceutically acceptable salt thereof. In someembodiments, the method comprises administering to the subject atherapeutically effective amount of (a) Compound 5 or a pharmaceuticallyacceptable salt thereof and (b) everolimus or a pharmaceuticallyacceptable salt thereof.

These and other features, together with the organization and manner ofoperation thereof, will become apparent from the following detaileddescription.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a graph showing the antitumor effect of multiple doses ofCompounds 1 and 3 on tumor volume in a TMD-8 lymphoma xenograft SCIDmouse model. “p.o., BID*14”: by mouth twice a day, for 14 days.

FIG. 1B is a graph showing the antitumor effect of Compounds 1 and 3 ontumor weight in the TMD-8 lymphoma xenograft SCID mouse model.

FIG. 2 is a graph showing the antitumor effect of Compound 3, Compound15, and their combination in the TMD-8 lymphoma xenograft SCID mousemodel.

FIG. 3 is a graph showing the antitumor effect of Compounds 3, 8, and15, and their combinations in the TMD-8 lymphoma xenograft SCID mousemodel.

FIG. 4 is a graph showing the antitumor effect of Compounds 3, 14, and16, and their combinations in the TMD-8 lymphoma xenograft SCID mousemodel.

FIG. 5 is a graph showing the antitumor effect of Compounds 3, 14, and15, and their combination in the TMD-8 lymphoma xenograft SCID mousemodel.

FIG. 6 is a graph showing the antitumor effect of Compounds 3, 14, and15, and their combinations in a DoHH-2 lymphoma xenograft SCID mousemodel.

FIG. 7 is a graph showing the antitumor effect of Compound 3, 14, and15, and their combination in the DoHH-2 lymphoma xenograft SCID mousemodel.

FIG. 8 is a graph showing the antitumor effect of Compounds 3 and 9 in aresistant WSU-DLCL2 SCID mouse model.

FIG. 9 is a graph showing the antitumor effect of Compounds 3, 14, and15, and their combination in the resistant WSU-DLCL2 SCID mouse model.

FIG. 10 is a graph showing the antitumor effect of triple combination ofCompounds 3, 14, and 15 at different dose combinations in the TMD-8lymphoma xenograft SCID mouse model.

FIG. 11 is a graph showing the antitumor effect of triple combinationsof Compound 3 or 9 with Compounds 14 and 15 in the DoHH2 mouse model.

FIG. 12 is a graph showing the antitumor effect of single, double, andtriple combination of Compound 3, Compound 12, and Compound 14 orCompound 8 at different dose combinations in the TMD-8 mouse model.

FIG. 13 is a graph showing the paw volume of the test animals in theAdjuvant-Induced Arthritis (AIA) study. Data are shown as mean±SEM.

FIG. 14 is a panel of representative photographic images of H&E stainingfrom each group of the AIA Study.

FIG. 15 is a graph showing the Clinical Score of the Collagen-InducedArthritis (CIA) Study. Data are shown as mean±SEM.

FIG. 16 is a panel of representative photographic images of H&E stainingfrom each group (40×) of the CIA Study.

DETAILED DESCRIPTION OF THE INVENTION

Signaling transduction pathways controlling cell growth, proliferation,survival, and apoptosis are complex and interrelated. Applicant hasdiscovered that concurrent blockade of (i) the BTK-mediated signalingpathway, (ii) the mTOR kinase-mediated signaling pathway, and (iii) theBcl-2-mediated signaling pathway or a signal transduction pathwaytargeted by an immunomodulatory drug (IMiD) provides surprisinglysuperior efficacy in treating cancer, such as hematologicalmalignancies, involving BTK, as compared to monotherapy targeting onlyone of these pathways.

Applicant has also discovered that concurrent blockade of (i) theBTK-mediated signaling pathway and (ii) the mTOR kinase-mediatedsignaling pathway provides surprisingly superior efficacy in treatingimmune disorders, such as autoimmune diseases, inflammation, andhypersensitivity, involving BTK, as compared to monotherapy targetingonly either pathway.

In one aspect of the invention, applicant has discovered the unexpectedstrong synergistic effects in the combined use of BTK, mTOR, and Bcl-2inhibitors to target the signal transduction mediated by these threecellular proteins. The relationships between signaling pathways arehighly complicated. See, e.g., Roschewski et al., “Diffuse large B-celllymphoma—treatment approaches in the molecular era,” Nat Rev Clin Oncol,2014, 11(1): 12-23, the content of which is herein incorporated byreference in its entirety. The super synergistic effects shown by thepresent combinations are surprising because not all combinations ofanti-cancer drugs have synergistic effects, much less synergisticeffects of the magnitude seen with the present combinations. Forexample, applicant has found that ALK inhibitor ceritinib and BTKinhibitor ibrutinib have no synergistic effect; applicant has also foundthat JAK1/2 inhibitor ruxolitinib and a BTK inhibitor, or an mTORinhibitor, or an immunomodulatory drug have no synergistic effect.

However, the “two in one” and “three in one” combination therapies ofthis invention have been found to be synergistic and superior tomonotherapy. The “two in one” pharmaceutical combinations each comprisethe use of:

(i) a BTK inhibitor and (ii) an mTOR kinase inhibitor;

(i) a BTK inhibitor and (ii) an IMiD;

(i) a BTK inhibitor and (ii) a TOPK inhibitor;

(i) a BTK inhibitor and (ii) a PI3K inhibitor; or

(i) a TOPK inhibitor and (ii) a PI3K inhibitors.

The “three in one” pharmaceutical combinations each comprise the use of:

(i) a BTK inhibitor, (ii) an mTOR kinase inhibitor, and (iii) animmunomodulatory drug (IMiD);

(i) a BTK inhibitor, (ii) an mTOR kinase inhibitor, and (iii) a Bcl-2inhibitor; or

(3) a BTK inhibitor, (ii) a PI3K inhibitor, and (iii) a Bcl-2 inhibitor.

Each of these inhibitors is further described below.

Single targeted therapy (monotherapy) requires longer term treatment andoften results in drug resistance and disease recurrence over time due togene mutations in the target (e.g., cancerous) cells. Indeed, resistanceto BTK inhibitor ibrutinib has been observed in patients. Thecombination therapies of this invention circumvent drug resistancebecause they inhibit potential compensatory pathways in the targetcells. The present invention thus brings new hopes to patients withrefractory diseases such as drug-resistant cancer. These therapies alsohave better safety profiles and broader therapeutic windows than singletargeted drugs, because the synergistic effects of the drugs in thesecombination therapies enable a healthcare provider to use the individualdrugs at much lower doses, thus reducing side effects of these drugs.

Applicant's studies have showed that the combination therapies of thisinvention achieved therapeutic efficacy that is as much as 100 timeshigher than monotherapy, yet at lower individual drug dosages. Invarious xenograft mouse models, oral gavage administration of acombination of 1/18 Compound 3 (BTK inhibitor)+1/6 everolimus (mTORinhibitor)+1/6 pomalidomide (IMiD) achieved far better therapeuticeffects than each of the three drugs at full doses. A “two in one”combination led to complete tumor regression in the 15-day treatmentcycle, and a “three in one” combination led to complete tumor regressionin an even shorter treatment cycle (9 days) in the mouse models.Importantly, the tumor did not rebound within 12 days after terminationof the treatment, unlike in single targeted therapy.

Applicant has found that the “three in one” pharmaceutical compositionscan inhibit up to 95% of tumor cell viability at a BTK inhibitorconcentration as low as 10 nM after an incubation time of only 48 hours,and that percentage increases with a longer incubation time (e.g., 72-96hours). Applicant has also shown that the inhibition of cell viabilityin vitro by the compositions correlates with the inhibition of tumorgrowth in vivo by them. It is thus expected that the presentcompositions can lead to cancer remissions or complete disappearance atan individual drug concentration as low as 10 nM. At present, singletargeted therapy or two-pathway combination therapy is effective ininhibiting the growth of tumor cells at the drug concentration of 1,000nM. For example, venetoclax, a Bcl-2 inhibitor, inhibited TMD-8 tumorcell viability at 1,000 nM, 100 nM and 10 nM, by 37.6%, 18.8% and 11.1%,respectively. A “two in one” pharmaceutical composition of thisinvention comprised of venetoclax at the same concentrations andCompound 3 (a BTK inhibitor; infra) at 1,000 nM, 100 nM, and 10 nMinhibited TMD-8 cell viability by 85.97%, 79.99% and 65.36%,respectively. A “three in one” pharmaceutical composition comprised ofvenetoclax at 100 nM, Compound 3 at 1,000 nM, 100 nM, and 10 nM, andPI3K inhibitor at 100 nM inhibited TMD-8 cell viability by 95.56%,95.30% and 94.62%, respectively. A “three in one” pharmaceuticalcomposition comprised of venetoclax at 100 nM, Compound 3 1,000 nM, 100nM and 10 nM and mTOR inhibitor everolimus at 100 nM inhibited TMD-8cell viability by 93.44%, 94.73% and 94.65%, respectively. Thesignificant synergistic effects of the compositions of this inventioncannot be inferred from the existing knowledge.

The present combination therapies have been shown not only highlyeffective in the sensitive TMD-8 tumor model, but also highly effectivein the insensitive DoHH2 tumor model and the resistant and refractoryWSU-DLCL tumor model. For example, compound EZ-6438 (histonemethyltransferase EZH2 inhibitor) inhibited tumor growth in the WSU-DLCLtumor model by oral gavage to mice at the high dose of 480 mg/kg/dayaccording to previous reports (Knutson et al., “Selective inhibition ofEZH2 by EPZ-6438 leads to potent antitumor activity in EZH2-mutantnon-Hodgkin lymphoma,” Mol. Can. Ther, 2014, 13(4):842-54), but the“three in one” combination of this invention is effective at only 21mg/kg/day for all three drugs combined.

In certain preferred embodiments, the pharmaceutical combinations of theinvention comprise (i) a BTK inhibitor, (ii) an mTOR kinase inhibitor,and (iii) an IMiD or a Bcl-2 inhibitor. In in vivo animal models, wherethe drugs were administered by oral gavage (a route of administrationthat can avoid uncertainties in pharmacokinetics and more accuratelyevaluate druggability of an oral pharmaceutical composition than, forexample, intraperitoneal injection or intravenous injection), such“three in one” combinations resulted in the complete disappearance oftumor grafts, and no tumor rebound was observed 12 days after treatmentended. Other combinations only inhibited tumor growth, and had to beadministered continuously to keep tumor growth at bay. Everolimusmonotherapy could result in the complete disappearance of tumor graftsat high doses (3 mg/kg), but tumors quickly rebounded after treatmentended. Thus, the “three in one” pharmaceutical combinations of thepresent invention not only cause total tumor regression, but it alsoprevents tumor recurrence.

The individual drugs useful in the present combination therapies aredescribed in further detail below.

BTK Inhibitors

BTK inhibitors useful in the present invention can be those known in theart, including but not limited to

-   -   ibrutinib,    -   acalabrutinib,    -   BGB-3111 (BeiGene),    -   spebrutinib,    -   ONO-4059 (Ono Pharmaceutical),    -   HM71224 (Hanmi Pharmaceutical),    -   RN486 (Roche),    -   4-(4-((4-((3-acrylamidophenyl)amino)-5-fluoropyrimidin-2-yl)amino)phenoxy)-N-methylpicolinamide        (CNX-774), and    -   N-[3-[4,5-dihydro-4-methyl-6-[[4-(4-morpholinylcarbonyl)phenyl]amino]-5-oxopyrazinyl]-2-methylphenyl]-4-(1,1-dimethylethyl)-benzamide        (CGI-1746).        They also can be the polyfluorinated compounds described in PCT        Publication WO 2015/165279 and U.S. application Ser. No.        15/075,033, filed Mar. 18, 2016, the disclosure of which are        incorporated by reference herein in their entireties. Besides        small molecules, other chemical entities, such as antisense        RNAs, siRNAs, peptidyl inhibitors and antibody inhibitors of BTK        can also be used. In some embodiments, the inhibitors bind        irreversibly to BTK. In some embodiments, the inhibitors can        bind to mutated BTK, such as BTK with a mutation at C481, e.g.,        a C481S mutation. In some embodiments, inhibitors of other        members of the BTK-mediated signaling pathway may be used in        lieu of or in addition to BTK inhibitors. For example,        inhibitors of Protein Kinase C (PKC) β such as enzastaurin and        sotrastaurin can be used as surrogates for a BTK inhibitor.

In certain embodiments, useful BTK inhibitors include those having astructure of one of the following formulae (Formulae I, II, Ia, Ib, IIa,and IIb) or their pharmaceutically acceptable salts thereof, and theirindividual enantiomers or diastereomers or salts thereof.

Nitrogen atom can form three bonds with other atoms. Any atom other thanhydrogen has to be drawn. Hydrogen may or may not be clearly drawn as atypical practice by chemists. For example, R—N means R—NH₂, R—NC(═O)—Wmeans R—NH(C═O)—W.

wherein:

each R¹ is F;

R² is F;

R³ is H or D;

n is 1, 2, 3 or 4; and

m is 1 or 2, or an enantiomer or diastereomer thereof, or apharmaceutically acceptable salt or prodrug thereof.

A compound of the above Formulae may comprise one or more stableisotopes or radio isotopes, including but not limited to, ²H, ³H, ¹³C,¹⁴C, ¹⁵N, and ¹⁸O. For example, ¹H, which is at the end of the doublebond of the vinyl group in the compound of Formula (I), maybe replacedwith ²H to reduce the drug inactivation caused by theoxidation/reduction of double bond.

As used herein, a “prodrug” is a biologically inactive compound that canbe metabolized in the body to produce a drug. For example, a prodrug ofa BTK inhibitor can be a prodrug at the amino group, for example, anamide, carbamate, or a polyethylene glycol.

As used herein, the term “pharmaceutically acceptable salts” refers tosalts formed with acid or base, including, but not limited to, (a) acidaddition salts: inorganic acid (e.g., hydrochloric acid, hydrobromicacid, sulfuric acid, phosphoric acid, nitric acid and other organicacids), and organic acid (e.g., acetic acid, oxalic acid, tartaric acid,succinic acid, malic acid, and ascorbic acid); and (b) base additionsalts, the formation of metal cations, such as zinc, calcium, sodium,and potassium.

Synthesis Schemes for BTK Inhibitors

A novel method for the synthesis of pyrazolopyrimidine compounds wassuccessfully designed. Representative synthesis schemes are shown below.Unless otherwise specified, in the following reaction schemes anddiscussion, R¹, R², R³, m, and n have the same meaning as defined above.

Fluoro-substituted starting material A1 is treated with substitutedphenol B1 to generate intermediate C1 under basic condition (e.g.,potassium carbonate) in a suitable solvent (e.g., DMF). Intermediate C1then reacts with bis(pinacolato)diboron to give intermediate D1 with asuitable catalyst (e.g.,[1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II)) under basiccondition (e.g., potassium acetate) in a suitable solvent (e.g.,1,4-dioxane). Iodination of 1H-pyrazolo[3,4-d]pyrimidin-4-amine with NISforms intermediate F1, followed by Mitsunobu reaction or displacementreaction to furnish intermediate G1. Intermediate G1 is treated withcompound D1 above obtained to give intermediate H1 with a suitablecatalyst (e.g., Pd-118) under basic condition (e.g., potassiumphosphate) in a suitable solvent (e.g., 1,4-dioxane). De-Boc protectionof intermediate H1 gives amine I1 under acidic condition. IntermediateI1 is reacted with an electrophilic reagent to form amide J1. If J1 isracemic, optically active compounds K1 and L1 can be obtained by SFCchiral resolution.

3-fluoro-4-bromophenol reacts with 1-fluoro-3-nitrobenzene to generateintermediate C2 with a base (e.g., potassium carbonate) in a suitablesolvent (e.g., DMF). The obtained nitro compound C2 is reduced to theamine D2 with appropriate reducing reagents (e.g., iron powder andammonium chloride) in appropriate solvents (e.g., ethanol and water),followed by treatment with sodium nitrite and hydrogen fluoride pyridineto generate fluoro-substituted intermediate E2. Intermediate E2 thenreacts with bis(pinacolato)diboron to give intermediate F2 with asuitable catalyst (e.g.,[1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II)) under basiccondition (e.g., potassium acetate) in a suitable solvent (e.g.,1,4-dioxane). Intermediate G1 is treated with compound F2 above obtainedto give intermediate G2 with a suitable catalyst (e.g., Pd-118) underbasic condition (e.g., potassium phosphate) in a suitable solvent (e.g.,1,4-dioxane). De-Boc protection of intermediate G2 gives amine H2 underacidic condition. Intermediate H2 is reacted with an electrophilicreagent to form amide 12. If 12 is racemic, optically active compoundsJ2 and K2 can be obtained by SFC chiral resolution.

3-fluoro-4-bromophenol reacts with 3-fluorophenylboronic acid togenerate intermediate B3 with an appropriate catalyst (e.g., copperacetate). Intermediate B3 then reacts with bis(pinacolato)diboron togive intermediate C3 with a suitable catalyst (e.g.,[1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II)).Intermediate G1 is treated with compound C3 above obtained to giveintermediate D3 with an appropriate catalyst (e.g.,[1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II)) under basiccondition (e.g., potassium acetate) in a suitable solvent (e.g.,1,4-dioxane). De-Boc protection of intermediate D3 gives amine E3 underacidic condition. Intermediate E3 is reacted with an electrophilicreagent to form amide F3. If F3 is racemic, optically active compoundsH3 and 13 can be obtained by SFC chiral resolution.

Amine I1 was reacted with but-2-ynoic acid to form amide II. If II isracemic, optically active compounds H3 and 13 could be obtained by SFCchiral resolution.

Intermediate A5 was formed via Mitsunobu reaction or displacementreaction from compound F1. A5 was treated with compound D1 aboveobtained to give intermediate B5 with a suitable catalyst (e.g., Pd-118)under basic condition (e.g., potassium phosphate) in a suitable solvent(e.g., 1,4-dioxane). De-Boc protection of intermediate B5 gave amine C5under acidic condition. Intermediate C5 was reacted with anelectrophilic reagent to form amide Ia.

Amine I1 was reacted with but-2-ynoic acid to form amide IIa.

Table 1 shows the structures and names of these compounds and Compounds7 and 20.

TABLE 1 Representative Compounds Compound No. Structure Name M + 1  1

1-((R)-3-(4-amino-3-(2-fluoro-4-(2,3,5,6-tetrafluorophenoxy)phenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)prop-2-en-1-one 531  2

1-((S)-3-(4-amino-3-(2-fluoro-4-(2,3,5,6-tetrafluorophenoxy)phenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)prop-2-en-1-one 531  3

1-((R)-3-(4-amino-3-(2-fluoro-4-(2,3,5,6-tetrafluorophenoxy)phenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyrrolidin-1-yl)prop-2-en-1-one 517  4

1-((S)-3-(4-amino-3-(2-fluoro-4-(2,3,5,6-tetrafluorophenoxy)phenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyrrolidin-1-yl)prop-2-en-1-one 517  5

1-((R)-3-(4-amino-3-(2-fluoro-4-(3-fluorophenoxy)phenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyrrolidin-1-yl)prop-2-en-1-one 463  6

(E)-1-((R)-3-(4-amino-3-(2-fluoro-4-(3-fluorophenoxy)phenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyrrolidin-1-yl)-3-deuterium- prop-2-en-1-one 518  7

(Z)-1-((R)-3-(4-amino-3-(2-fluoro-4-(3-fluorophenoxy)phenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyrrolidin-1-yl)-3-deuterium- prop-2-en-1-one 518 20

1-((R)-3-(4-amino-3-(2-fluoro-4-(3-fluorophenoxy)phenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyrrolidin-1-yl)but-2-yn-1-one 475 Note: If there aredifferences between the structure and name, the structure will prevail.

Pharmacokinetic analysis of the Compounds can be performed as descriedin Marostica et al., “Population pharmacokinetic model of ibrutinib, aBruton tyrosine kinase inhibitor, in patients with B cell malignancies,”Cancer Chemother Pharmacol, 2015, 75:111-121. The content of thispublication is herein incorporated by reference in its entirety.

Toxicity and toxicokinetic (TK) studies of the compounds can beperformed by well known methods. Applicant's TK studies showed that theBTK-inhibitory Compounds described herein had better safety profilesthan ibrutinib in 28-day rat and dog studies. For example, Compound 3demonstrated the following advantageous characteristics:

(i) higher no-observed-adverse-effect-level (NOAEL) than ibrutinib;

(ii) 5- to 14-fold higher exposure than ibrutinib at the same dose of 40mg/kg in rats on day 1;

(iii) when administered to rats at 40 mg/kg, AUC (area under the curve)13,700 h*ng/mL (male) and 17,300 h*ng/mL (female), as compared to 1,000h*ng/mL (male) and 3300 h*ng/mL (female) for ibrutinib at 40 mg/kg(according to U.S. FDA NDA Application No. 205552Orig1s000_pharmacological review);

(iv) when administered to dogs at 15 mg/kg, AUC 3,550 (male) and 2,930(female) h*ng/mL, as compared to AUC 1,780 (male) and 1,850 (female)h*ng/mL for ibrutinib at 24 mg/kg ((according to U.S. FDA NDAApplication No. 205552Orig1s000_ pharmacological review);

(v) no significant difference in drug exposure between Day 1 and Day 28;and

(vi) no significant difference in drug exposure between male and female.

These characteristics show that Compound 3 has low toxicity, excellentpharmacokinetics, and superior bioavailability when compared toibrutinib.

mTOR Kinase Inhibitors

The mammalian target of rapamycin (mTOR) is a protein kinase that servesas a key regulator of cell growth, proliferation, metabolism andapoptosis. Inhibitors of mTOR kinase useful in the combination therapyof this invention include but are not limited to

-   -   everolimus,    -   rapamycin,    -   [7-(6-Amino-3-pyridinyl)-2,3-dihydro-1,4-benzoxazepin-4(5H)-yl][3-fluoro-2-methyl-4-(methylsulfonyl)phenyl]-methanone        (XL388),    -   N-ethyl-N′-[4-[5,6,7,8-tetrahydro-4-[(3S)-3-methyl-4-morpholinyl]-7-(3-oxetanyl)pyrido[3,4-d]pyrimidin-2-yl]phenyl]-Urea        (GDC-0349),    -   3-(2,4-bis((S)-3-methylmorpholino)pyrido[2,3-d]pyrimidin-7-yl)-N-methylbenzamide        (AZD2014),    -   (5-(2,4-bis((S)-3-methylmorpholino)pyrido[2,3-d]pyrimidin-7-yl)-2-methoxyphenyl)methanol        (AZD8055),    -   GSK105965,    -   3-(2-aminobenzo[d]oxazol-5-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine        (MLNO 128),    -   temsirolimus,    -   ridaforolimus,    -   PI-103,    -   NVP-BEZ23 5,    -   WJD008,    -   XL765,    -   SF-1126,    -   Torin1,    -   PP242,    -   PP30,    -   Ku-0063794,    -   WYE-354,    -   WYE-687,    -   WAY-600,    -   INK128,    -   OSI-027, and    -   pharmaceutically acceptable salts thereof.

In some embodiments, everolimus may be preferred. Everolimus has beenapproved by the United States Food and Drug Administration for thetreatment of breast cancer, pancreatic cancer, renal cell carcinoma,renal angiomyolipoma, and tuberous sclerosis. In addition, everolimushas been used to treat organ transplant rejection at low doses, as organtransplant also activates mTOR. Applicant contemplates that thecombination therapy of this invention also can be used in thesecontexts.

Besides small molecules, other chemical entities, such as antisenseRNAs, siRNAs, peptidyl inhibitors and antibody inhibitors of mTOR canalso be used. Further, inhibitors of other members of the mTOR-mediatedsignaling pathway may be used in lieu of or in addition to mTORinhibitors. For example, inhibitors of phosphoinositide 3-kinase (PI3K)such as BTG226, gedatolisib, apitolisib, omipalisib, dactolisib,duvelisib, and idelalisib can be used in lieu of or in addition to mTORinhibitors. Inhibitors of Akt (Protein Kinase B) such as8-[4-(1-aminocyclobutyl)phenyl]-9-phenyl-2H-[1,2,4]triazolo[3,4-f][1,6]naphthyridin-3-one;dihydrochloride (MK-2206) also can be used in lieu of or in addition tomTOR inhibitors.

Immunomodulatory Drugs

Immunomodulatory drugs (IMiDs) are a class of drugs that includethalidomide and its structural and functional analogues. IMiDs possessanti-angiogenic, anti-proliferative and pro-apoptotic properties forcancer cells. IMiDs stimulate T lymphocytes to induce proliferation,cytokine production, and cytotoxicity, thus increasing T cells'anti-cancer activities. IMiDs are useful in treating a variety ofinflammatory and autoimmune diseases. IMiDs also are useful in treatingneoplastic diseases such as hematologic neoplasms, e.g., multiplemyeloma and myelodysplastic syndromes, as well as certain solid tumors.IMiDs such as lenalidomide, pomalidomide, CC-112 (Celgene), and CC-220(Celgene) have improved potency and reduced side effects compared tothalidomide.

Bcl-2 Inhibitors

B-cell lymphoma 2 protein (Bcl-2) is an important regulator ofprogrammed cell death (apoptosis). Bcl-2 inhibitors useful in thisinvention include, but are not limited to:

-   -   venetoclax,    -   sabutoclax,    -   navitoclax,    -   obatoclax,    -   4-[4-[[2-(4-Chlorophenyl)phenyl]methyl]piperazin-1-yl]-N-[4-[[(2R)-4-(dimethylamino)-1-phenylsulfanylbutan-2-yl]amino]-3-nitrophenyl]sulfonylbenzamide        (ABT-737),        N-[4-(2-tert-butylphenyl)sulfonylphenyl]-2,3,4-trihydroxy-5-[(2-propan-2-ylphenyl)methyl]benzamide        (TW-37), and    -   pharmaceutically acceptable salts thereof.        Besides small molecules, other chemical entities, such as        antisense RNAs, siRNAs, peptidyl inhibitors and antibody        inhibitors of Bcl-2 can also be used. Further, inhibitors of        other members of the Bcl-2-mediated signaling pathway may be        used in lieu of or in addition to Bcl-2 inhibitors.        Additional Combinations

The combination therapy of this invention may be further combined withother therapeutic agents, such as a TOPK inhibitor (e.g., OTS964((R)-9-(4-(1-(dimethylamino)propan-2-yl)phenyl)-8-hydroxy-6-methylthieno[2,3-c]quinolin-4(5H)-one) (Oncotherapy Science)), another tyrosine kinaseinhibitor (e.g., axitinib, dasatinib, icotinib), a topoisomeraseinhibitor (e.g., topotecan), a sphingosine-1-phosphate receptor agonist(e.g., fingolimod, KRP-203), anti-T cell immunoglobulin (e.g., AtGam),anti-IL-2 receptor antibody (e.g., daclizumab), amides (CTX), ifosfamide(IFO), adriamycin (ADM), daunorubicin (DNR), vincristine (VCR),vinblastine (VBL), etoposide (VP16), vermeer (Vumon), carboplatin (CBP)and methotrexate (MTX) cyclosporin A, tacrolimus, sirolimus, everolimus,azathioprine, brequinar, leflunomide, LEA-29Y, anti-CD3 antibody (e.g.,OKT3), aspirin, B7-CD28 blocking molecules (e.g., belatacept,abatacept), CD40-CD154 blocking molecules (anti-CD40 antibodies),acetaminophen, ibuprofen, naproxen, piroxicam, and anti-inflammatorysteroids (e.g., prednisolone or dexamethasone).

Diseases

The combination therapies of this invention can treat a variety ofconditions in which BTK inhibition is beneficial. These conditionsinclude, without limitation (1) autoimmune diseases, such as chroniclymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetesmellitus, myasthenia gravis, chronic ulcerative colitis, ulcerativecolitis, Crohn's disease, inflammatory bowel disease, pernicious anemiaassociated with chronic atrophic gastritis, Goodpasture's syndrome,pemphigus vulgaris, pemphigoid, primary biliary cirrhosis, multiplecerebrospinal sclerosis, acute idiopathic neuritis, systemic lupuserythematosus, rheumatoid arthritis, psoriasis, systemic vasculitis,scleroderma, pemphigus, mixed connective tissue disease, multiplesclerosis, autoimmune hemolytic anemia, and autoimmune thyroid disease;(2) hypersensitivity diseases, such as serum sickness, asthma, allergicrhinitis, drug allergy; and (3) inflammatory diseases, such askeratitis, rhinitis, stomatitis, mumps, pharyngitis, tonsillitis,tracheitis, bronchitis, pneumonia, myocarditis, gastritis,gastroenteritis, cholecystitis, and appendicitis. The therapies may alsobe used in treating rejection in transplantation.

The combination therapies of this invention can also be used to treat avariety of cancer, including hematological malignancies such as B-cellmalignancies, e.g., small lymphocytic lymphoma (SLL), prolymphocyticleukemia (PLL), acute lymphocytic leukemia (ALL), chronic lymphocyticleukemia (CLL), Richter's syndrome, diffuse large B-cell lymphoma(DLBCL), Waldenström Macroglobulinemia (WM), follicular lymphoma (FL),multiple myeloma, mantle cell lymphoma (MCL)), marginal zone lymphoma,Hodgkin lymphoma, and non-Hodgkin lymphoma.

In some embodiments, the combination therapies of this invention is usedas a first line therapy, to treat patients who have not been treated byanother drug for the same condition. In other embodiments, thecombination therapy of this invention is used as a second, third, orfourth line therapy, where the patients have been treated for the samecondition unsuccessfully (e.g., refractory or relapsed) by another drug,for example, rituximab (which targets CD20 on B cells), CHOP (thecyclophosphamide-hydroxydaunorubicin-oncovin-prednisone therapy), orrituximab plus CHOP (R-CHOP).

Pharmaceutical Compositions and Administration

The individual drugs in the combination therapies of the presentinvention can be administered separately to the patient, in any order asdeemed appropriate for the patient by the healthcare provider. They canalso be administered simultaneously; or in a hybrid manner, that is, forexample, two of the individual drugs are administered simultaneously,separately from a third drug.

The individual drugs in the combination therapies can also beco-formulated or provided in a pharmaceutical kit. In some embodiments,the co-formulated pharmaceutical composition or the pharmaceutical kitcomprises a BTK inhibitor, an mTOR inhibitor, and an IMiD as activeingredients. In other embodiments, the co-formulated pharmaceuticalcomposition or the pharmaceutical kit comprises a BTK inhibitor, an mTORinhibitor, and a Bcl-2 inhibitor as active ingredients. In otherembodiments, the co-formulated pharmaceutical composition or thepharmaceutical kit comprises a BTK inhibitor, a PI3K inhibitor, and aBcl-2 inhibitor as active ingredients. In other embodiments, theco-formulated pharmaceutical composition or the pharmaceutical kitcomprises a BTK inhibitor, a PI3K inhibitor, and an IMiD as activeingredients. In some embodiments, the co-formulated pharmaceuticalcomposition or the pharmaceutical kit comprises two or three compoundsselected from BTK inhibitors, mTOR kinase inhibitors, IMiDs, Bcl-2inhibitors, and PI3K inhibitors as active ingredients.

Also included in the invention are the aforementioned combinations ofactive ingredients for use in treating diseases where BTK inhibition arebeneficial, including, without limitation cancer such as lymphoidmalignancies (e.g., B-cell malignancies recited above), and immunedisorders such as autoimmune diseases and inflammation. Further includedin the invention is the use of the aforementioned combinations of activeingredients in the manufacture of medicament for the treatment of thesediseases.

Carriers, excipients and other additives commonly used forpharmaceutical preparations may be used to prepare pharmaceuticalcompositions containing the active ingredients of the present invention,or pharmaceutically acceptable salts thereof.

The administration forms may be oral dosage forms, such as tablets,pills, capsules, granules, powders, emulsions, syrups, suspensions,liquid preparations; or non-oral dosage forms, such as forms forintravenous, subcutaneous or intramuscular injection, suppository,transdermal implant, or inhalation. Symptoms, age, gender, weight, andother relevant medical information of the patient should be consideredin order to properly determine the dosage of the drugs. Generallyspeaking, for oral administration, daily doses for adult patients of adrug is about 0.001 mg/kg to 100 mg/kg, given in a single dose daily ordivided into 2 to 4 subdoses daily; for intravenous administration,daily doses for adult patients is 0.0001 mg/kg to 10 mg/kg, administeredonce or more times daily.

In the present invention, solid compositions for oral administration maybe tablets, capsules, powders, granules and the like. In such solidcompositions, one or more active substances with at least one inertexcipient (e.g., lactose, mannitol, glucose, hydroxypropylcellulose,microcrystalline cellulose, starch, poly vinyl pyrrolidone, magnesiumaluminum silicate, and the like) can be mixed. The compositions maycontain inert additives such as lubricants (e.g., magnesium stearate),disintegrating agents (e.g., sodium carboxymethyl starch) anddissolution aids. If necessary, tablets or pills may be coated withappropriate coatings such as a sugar coating or a gastric or entericcoating agent.

The liquid compositions for oral administration include pharmaceuticallyacceptable emulsions, solutions, aqueous or oily suspensions, syrups,elixirs, and commonly used inert diluent (e.g., purified water, andethanol). In addition to the inert diluent, the composition may alsocontain additives such as solubilizing agents, wetting agents,suspending agents, and sweetener, flavoring agents, flavoring agents andpreservatives.

Injections for parenteral administration may include sterile aqueous ornon-aqueous liquid preparations, suspensions, and emulsions. Diluentaqueous solutions may include distilled water and physiological saline.Non-aqueous diluent solutions may include propylene glycol, polyethyleneglycol, vegetable oils, alcohols (e.g., ethanol), and polysorbate 80.Such compositions may further contain isotonic agents, such aspreservatives, wetting agents, emulsifying agents, dispersing agents,stabilizing agents, dissolving aids and the like. The compositions canbe sterilized by filtration through a bacteria retaining filter,addition of bactericides, or irradiation. In addition, thesecompositions may be made as sterile solid compositions and dissolved orsuspended in sterile water or a sterile solvent for injection prior touse.

Pharmaceutical compositions used for transmucosal administration such asinhalation and nasal absorption can be solid, liquid, or semi-solidstate of use, and can be made in accordance with conventional methods.For example, excipients such as lactose, starch, pH adjusting agents,preservatives, surfactants, lubricants, stabilizing and thickeningagents and the like can be added. A suitable inhalation or insufflationdevice can be used. For example, metered dose inhaler devices may beused. A pressurized aerosol spray can also be used with a suitablepropellant (e.g., chlorofluoroalkane, hydrofluoroalkane, or a suitablegas such as carbon dioxide).

The following examples are meant to illustrate the methods and materialsof the present invention. Suitable modifications and adaptations of thedescribed conditions and parameters normally encountered in the artwhich are obvious to those skilled in the art are within the spirit andscope of the present invention.

WORKING EXAMPLES Example 1 Synthesis of BTK Inhibitors

Compound 1 and Compound 2

Step A:

Potassium carbonate (68.0 g, 492.1 mmol, 2.0 eq.) and the compound1,2,3,4,5-pentafluorophenyl (49.6 g, 295.3 mmol, 1.2 eq.) was added to asolution of 3-fluoro-4-bromophenol (47.0 g, 246.1 mmol, 1.0 eq.) in DMF(500 mL). The reaction was stirred at 100° C. for 12 hours. Solvent wasremoved under reduced pressure. The residue was dissolved in ethylacetate (300 mL), washed with water (100 mL) and brine (100 mL×2). Theorganic phase was dried over anhydrous sodium sulfate, and concentratedto give the title compound (78 g, yield: 93%).

Step B:

3-(4-bromo-3-fluorophenoxy)-1,2,4,5-tetrafluorobenzene (73 g, 215.3mmol, 1.0 eq.), bis pinacolato boronate (65.6 g, 258.4 mmol, 1.2 eq.),potassium acetate (31.6 g, 322.9 mmol, 1.5 eq.) and (dppf)PdCl₂ (9.4 g,12.8 mmol, 0.06 eq.) were added to 1,4-dioxane (1 L). The resultingmixture was stirred at 80° C. for 14 hours under nitrogen. After coolingto room temperature, the reaction mixture was filtered through Celite.The filtrate was concentrated to give the crude product, which waspurified by silica gel column chromatography (eluent: petroleum ether)to give the title compound (60 g, yield: 72%).

Step C:

NIS (250 g, 1.11 mol, 1.5 eq.) was added to a solution of1H-pyrazolo[3,4-d]pyrimidin-4-amine (100 g, 0.74 mol, 1.0 eq.) in DMF(800 mL). The reaction was stirred at 80-85° C. for 16 hours undernitrogen. The reaction mixture was filtered. The filter cake was washedwith ethanol (1000 mL×3) to give the title compound (184 g, yield: 95%).

Step D:

Triethylamine (15 g, 150 mmol, 3.0 eq.) and methanesulfonyl chloride(6.3 g, 55 mmol, 1.1 eq.) were sequentially added dropwise to a solutionof 3-hydroxy-piperidine-1-carboxylate (10.0 g, 50 mmol, 1.0 eq.) indichloromethane (100 mL) at 0° C. The reaction was stirred at 20° C. for1 hour, and then quenched with saturated NaHCO₃ (100 mL). The resultingmixture was extracted with dichloromethane (200 mL×3). The combinedorganic phases were dried over anhydrous sodium sulfate, andconcentrated under reduced pressure to give the title compound (13 g,yield: 95%).

Step E:

cesium carbonate (20.2 g, 62 mmol, 2.0 eq.) and 3-(methylsulfonyloxy)piperidine-1-carboxylate (13 g, 46.5 mmol, 1.5 eq.) was added to asolution of 3-iodo-1H-pyrazolo[3,4-d]-pyrimidin-4-amine (8.1 g, 31 mmol,1.0 eq) in DMF (50 mL) at 0° C. The reaction was stirred at 80° C.overnight. After cooling to room temperature, the mixture was filteredthrough Celite, and concentrated under reduced pressure to give thecrude product, which was purified by silica gel column chromatography(eluent: ethyl acetate) to give the title compound (5 g, yield: 25%).

Step F:

tert-butyl3-(4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidine-1-carboxylate(7.6 g, 17.1 mmol, 1.0 eq.),2-(2-fluoro-4-(2,3,5,6-tetrafluorophenoxy)phenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane(8.6 g, 22.3 mmol, 1.3 eq.), potassium phosphate (7.3 g, 34.2 mmol, 2.0eq.) and Pd-118 (0.56 g, 0.855 mmol, 0.05 eq.) were added to a mixtureof 1,4-dioxane/water (5/1, v/v, 240 mL). The reaction stirred at 60° C.for 12 hours under nitrogen atmosphere. After cooling to roomtemperature, the reaction mixture was poured into ice water (300 mL) andthen extracted with ethyl acetate (100 mL×4). The combined organicphases were dried over anhydrous sodium sulfate, and concentrated togive the crude product, which was purified by silica gel columnchromatography separation (eluent: ethyl acetate) to give the titlecompound (6.8 g, yield: 69%).

Step G:

HCl/EtOAc (20 mL, 4 mol/L) was added to a solution of tert-butyl3-(4-amino-3-(2-fluoro-4-(2,3,5,6-tetrafluorophenoxy)phenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidine-1-carboxylate(6.8 g, 11.8 mmol) in ethyl acetate (50 mL) at 0° C. The reaction wasstirred at room temperature for 1 hour, and then concentrated to givethe title compound hydrochloride (5.2 g, yield: 86%).

Step H:

Triethylamine (887 mg, 8.7 mmol, 3.0 eq.) and acryloyl chloride (0.26 g,2.9 mmol, 1.0 eq.) were sequentially added dropwise to a solution of3-(2-fluoro-4-(2,3,5,6-tetrafluorophenoxy)phenyl)-1-(piperidin-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine(1.5 g, 2.9 mmol, 1.0 eq.) in dichloromethane (10 mL) at 0° C. Thereaction was stirred at 0° C. for 1 hour, quenched with water (5 mL),diluted with dichloromethane (50 mL), and washed with water (30 mL×2)and saturated brine (30 mL). The organic phase was dried over anhydroussodium sulfate and concentrated to give the crude product, which waspurified by silica gel column chromatography to give the title compound(eluent: petroleum ether:ethyl acetate=1:0˜1:1) (0.94 g, yield: 64%).

LC/MS (method: UFLC): RT=3.130 min; m/z=531.1 [M+H]⁺; Total runningtime=7.000 min.

¹H NMR (400 MHz, DMSO-d₆) δ 8.22 (s, 1H), 8.00-7.91 (m, 1H), 7.55-7.46(m, 1H), 7.27 (dd, J=2.4, 10.8 Hz, 1H), 7.12 (dd, J=2.4, 8.8 Hz, 1H),6.88-6.65 (m, 1H), 6.13-6.02 (m, 1H), 5.70-5.56 (m, 1H), 4.71-4.65 (m,1H), 4.54-4.51 (m, 0.5H), 4.20-4.17 (m, 1H), 4.07-4.04 (m, 0.5H),3.67-3.60 (m, 0.5H), 3.17-3.12 (m, 1H), 2.98-2.94 (m, 0.5H), 2.26-2.21(m, 1H), 2.11-2.06 (m, 1H), 1.92-1.89 (m, 1H), 1.58-1.54 (m, 1H).

Step I:

Racemate1-(3-(4-amino-3-(2-fluoro-4-(2,3,5,6-tetrafluorophenoxy)phenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)prop-2-en-1-one(750 mg) was separated by SFC chiral resolution (CO₂:C₂H₅OH(0.2% DEA),v/v, 200 ml/min) to give Compound 11-((R)-3-(4-amino-3-(2-fluoro-4-(2,3,5,6-tetrafluorophenoxy)phenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)prop-2-en-1-one(280 mg, ee: 100%) and Compound 21-((S)-3-(4-amino-3-(2-fluoro-4-(2,3,5,6-tetrafluorophenoxy)phenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)prop-2-en-1-one(330 mg, ee: 98%).

Compound 1:

LC/MS (method: UFLC): RT=3.002 min; m/z=531.1 [M+H]⁺; Total runningtime=7.000 min.

¹H NMR (400 MHz, CDCl₃) δ 8.36 (s, 1H), 7.58 (t, J=8.4 Hz, 1H),7.09-7.04 (m, 1H), 6.94-6.88 (m, 2H), 6.62-6.54 (m, 1H), 6.32-6.25 (m,1H), 5.73-5.63 (m, 1H), 5.56-5.51 (m, 1H), 4.90-4.85 (m, 1.5H),4.59-4.56 (m, 0.5H), 4.21-4.17 (m, 0.5H), 4.04-4.01 (m, 0.5H), 3.76-3.71(m, 0.5H), 3.40-3.35 (m, 0.5H), 3.22-3.15 (m, 0.5H), 2.93-2.87 (m,0.5H), 2.39-2.27 (m, 2H), 2.04-1.68 (m, 2H).

Compound 2:

LC/MS (method: UFLC): RT=3.006 min; m/z=531.1 [M+H]⁺; Total runningtime=7.000 min.

¹H NMR (400 MHz, CD₃OD) δ 8.24 (s, 1H), 7.62 (t, J=8.4 Hz, 1H),7.50-7.45 (m, 1H), 7.09-7.01 (m, 2H), 6.85-6.63 (m, 1H), 6.21-6.09 (m,1H), 5.77-5.61 (m, 1H), 4.63-4.59 (m, 1H), 4.23-4.07 (m, 1.5H),3.90-3.85 (m, 0.5H), 3.51-3.45 (m, 0.5H), 3.34-3.17 (m, 1.5H), 2.40-2.23(m, 2H), 2.08-2.05 (m, 1H), 1.75-1.71 (m, 1H).

Compound 3

Method 1:

Step A:

DIAD (27.6 g, 137.5 mmol, 1.5 eq.) was added dropwise to a mixture of3-iodo-1H-pyrazolo[3,4-d]pyrimidin-4-amine (24 g, 92 mmol, 1.0 eq.),(S)-tert-butyl 3-hydroxypyrrolidine-1-carboxylate (26 g, 137.5 mmol, 1.5eq.) and PPh₃ (36 g, 137.5 mmol, 1.5 eq.) in tetrahydrofuran (720 mL) at0° C. and under nitrogen atmosphere. The reaction was stirred at 0° C.for 1 hour, then stirred overnight at room temperature. After theremoval of solvent under reduced pressure, acetonitrile (200 mL) wasadded to residues. The mixture was stirred at room temperature for 2hours and filtered. The filter cake was washed with acetonitrile (20 mL)and dried to give the title compound (25 g, yield: 63%).

Step B:

(R)-tert-butyl3-(4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyrrolidine-1-carboxylate(25 g, 58 mmol, 1.0 eq.),2-(2-fluoro-4-(2,3,5,6-tetrafluorophenoxy)phenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane(30 g, 75.4 mmol, 1.3 eq.), potassium phosphate (25 g, 116 mmol, 2.0eq.) and Pd-118 (750 mg, 1.16 mmol, 0.02 eq.) were added to a mixture of1,4-dioxane/water (5/1, v/v, 600 mL). The reaction was stirred at 60° C.overnight under nitrogen atmosphere. After cooling to room temperature,the mixture was filtered through Celite. Filtrate was concentrated underreduced pressure. Water (300 mL) was added to the residue, thenextracted with ethyl acetate (300 mL×3). The combined organic phaseswere dried over anhydrous sodium sulfate, and concentrated to give thetitle compound (60 g, crude).

Step C:

HCl/EtOAc (100 mL, 4 mol/L) was added to a solution of (3R)-tert-butyl3-(4-amino-3-(2-fluoro-4-(2,3,5,6-tetrafluorophenoxy)phenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyrrolidine-1-carboxylate(60 g, crude) in ethyl acetate (100 mL) at 0° C. The reaction wasstirred at room temperature for 1 hour and concentrated to dryness togive the hydrochloride salt of the title compound. Water (500 mL) wasadded to the reaction flask, extracted with ethyl acetate (300 mL×3).The aqueous phase was adjusted pH=9, and then extracted with ethylacetate (300 mL×3). The combined organic phases were dried overanhydrous sodium sulfate, and concentrated under reduced pressure togive the title compound (24 g, two steps yield: 90%).

Step D:

NaOH (10%, 94 mL) was added to a solution of3-(2-fluoro-4-(2,3,5,6-tetrafluorophenoxy)phenyl)-1-((R)-pyrrolidin-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine(23.5 g, 50.75 mmol, 1.0 eq) in tetrahydrofuran (470 mL) at −5° C., andthen acryloyl chloride (5.97 g, 66 mmol, 1.3 eq.) was added dropwise.The reaction was stirred at −5° C. for 1 hour, quenched with saturatedbrine (100 mL) and extracted with ethyl acetate (200 mL×3). The combinedorganic phases were dried over anhydrous sodium sulfate, andconcentrated to give the crude product, which was purified by silica gelcolumn chromatography (eluent: petroleum ether:ethyl acetate=1:3˜1:1).The product obtained was dissolved in methanol (500 mL) and filtered.Water (1500 mL) was added to the stirred filtrate, stirred for 2 hoursand filtered. The filter cake was dried under reduced pressure to givethe title compound (16.5 g, yield: 63%).

LC/MS (method: UFLC): RT=3.764 min; m/z=517.0 [M+H]⁺; Total runningtime=7.000 min.

¹H NMR (400 MHz, CD₃OD) δ 8.45 (s, 1H), 7.70 (t, J=8.4 Hz, 1H),7.55-7.46 (m, 1H), 7.12-7.05 (m, 2H), 6.70-6.55 (m, 1H), 6.33-6.26 (m,1H), 5.81-5.75 (m, 1H), 4.23-3.83 (m, 5H), 2.68-2.55 (m, 2H).

Method 2:

NaOH (216 mg, 5.40 mmol, 2.5 eq.) was added to a solution of3-(2-fluoro-4-(2,3,5,6-tetrafluorophenoxy)phenyl)-1-((R)-pyrrolidin-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine(1.0 g, 2.16 mmol, 1.0 eq.) in tetrahydrofuran (50 mL) and water (10 mL)at 0° C., and then a solution of chloropropionyl chloride (288 mg, 2.27mmol, 1.05 eq.) in tetrahydrofuran (10 mL) was added dropwise. Thereaction was stirred at 0° C. for 1 hour, then at 60° C. for 12 hours.After cooling to room temperature, saturated brine (10 mL) was added,and then extracted with ethyl acetate (50 mL×3). The combined organicphases were dried over anhydrous sodium sulfate, and concentrated togive the crude product, which was purified by silica gel columnchromatography (eluent: petroleum ether:ethyl acetate=1:3˜1:1) to giveCompound 3 (0.8 g, yield: 71%).

Method 3:

(R)-1-(3-(4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyrrolidin-1-yl)prop-2-en-1-one(100 g, 0.26 mmol, 1.0 eq.),2-(2-fluoro-4-(2,3,5,6-tetrafluorophenoxy)phenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane(120 mg, 0.31 mmol, 1.2 eq.), sodium carbonate (55 mg, 0.52 mmol, 2.0eq.) and Pd (PPh₃)₄(30 mg, 0.026 mmol, 0.01 eq.) was added to a mixtureof 1,4-dioxane/water (5 mL, 1/1, v/v). The reaction was stirred undermicrowave irradiation at 80° C. for 30 minutes. After cooling to roomtemperature, reaction mixture was filtered through Celite. The filtratewas concentrated to give the crude product, which was purified by HPLCseparation on a (C18 column, mobile phase: acetonitrile/water/0.5% HCl,eluent gradient 10% to 100% (volume ratio)). After the removal ofvolatile solvent, the desired fraction was lyophilized to give the titlecompound (38 mg, yield: 28%).

Method 4:

Compound 3 and Compound 4

Step A:

Triethylamine (35 g, 346 mmol, 2.1 eq.) was added to a solution of3-hydroxypyrrolidine-1-carboxylate (30.0 g, 163 mmol, 1.0 eq.) indichloromethane (200 mL) at 0° C., and then methyl chloride (36.6 g, 321mmol, 1.9 eq.) was added dropwise. The reaction was stirred at 0° C. for3 hours, quenched with water (100 mL), washed with water (20 mL×2) andsaturated brine (100 mL). The organic phase was dried over anhydroussodium sulfate and concentrated to give the title compound (45.6 g,yield: 100%).

Step B:

Cesium carbonate (37 g, 115 mmol, 3.0 eq.) and the compound3-iodo-1H-pyrazolo[3,4-d]pyrimidin-4-amine (10 g, 38 mmol, 1.0 eq.) wereadded to a solution of tert-butyl3-(methylsulfonyloxy)pyrrolidine-1-carboxylate (35 g, 134 mmol, 3.5 eq.)in DMF (300 mL). The reaction was stirred at 85° C. for 12 h. Aftercooling to room temperature, the mixture was filtered. The filtrate wasconcentrated to give the crude product, which was purified by silica gelcolumn chromatography (eluent: petroleum ether:ethyl acetate=1:1) togive the title compound (7.0 g, yield: 44%).

Step C:

Tert-butyl3-(4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyrrolidine-1-carboxylate(8 g, 18 mmol, 1.0 eq.),2-(2-fluoro-4-(2,3,5,6-tetrafluorophenoxy)phenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane(10.7 g, 27 mmol, 1.5 eq.), potassium phosphate (7.6 g, 36 mmol, 2.0eq.) and Pd-118 (1.2 g, 1.8 mmol, 0.1 eq.) were added to a mixture of1,4-dioxane/water (180 mL, 5/1, v/v). The reaction under nitrogen andstirred at 60° C. for 14 hours. After cooling to room temperature, thereaction mixture was poured into ice water (50 mL) and extracted withethyl acetate (100 mL×3). The combined organic phases were dried overanhydrous sodium sulfate and concentrated to give the crude product,which was purified by silica gel column chromatography (eluent: ethylacetate:petroleum ether=1:1) to give the title compound (2.5 g, yield:25%).

Step D:

HCl/EtOAc (20 mL, 4 mol/L) was added to a solution of tert-butyl3-(4-amino-3-(2-fluoro-4-(2,3,5,6-tetrafluorophenoxy)phenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyrrolidine-1-carboxylate(2.5 g, 4.4 mmol) in dichloromethane (20 mL) at 0° C. The reaction wasstirred for 1 hour at room temperature, and then concentrated to underpressure give the title compound hydrochloride (2.2 g, yield: 100%).

Step E:

Triethylamine (1.4 g, 12.8 mmol, 3.0 eq.) was added to a solution of3-(2-fluoro-4-(2,3,5,6-tetrafluorophenoxy)phenyl)-1-(pyrrolidin-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine(2.2 g, 4.4 mmol, 1.0 eq.) in dichloromethane (50 mL), then acryloylchloride (0.38 g, 4.2 mmol, 0.95 eq.) was added dropwise at 0° C. Thereaction was stirred at 0° C. for 1 hour and quenched with water (30mL). The aqueous phase was extracted with methylene chloride (30 mL×3).The combined organic phases were dried over anhydrous sodium sulfate andconcentrated to give the crude product, which was purified by silica gelcolumn chromatography (eluent: ethyl acetate) to give the title compound(1.0 g, yield: 45%).

LC/MS (method: UFLC): RT=2.810 min; m/z=517.1 [M+H]⁺; Total runningtime=7.000 min.

Step F:

Racemate1-(3-(4-amino-3-(2-fluoro-4-(2,3,5,6-tetrafluorophenoxy)phenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyrrolidin-1-yl)prop-2-en-1-onewas separated by SFC chiral resolution to give Compound 3 (280 mg) andCompound 4 (320 mg).

Compound 4:

LC/MS (method: UFLC): RT=2.808 min; m/z=517.1 [M+H]⁺; Total runningtime=7.000 min.

Compound 5

Step A:

1-fluoro-3-nitrobenzene (29.6 g, 210 mmol, 1.0 eq.) and potassiumcarbonate (58 g, 420 mmol, 2.0 eq.) were added to a solution of3-fluoro-4-bromophenol (40 g, 210 mmol, 1.0 eq.) in DMF (400 mL). Thereaction was stirred at 90° C. for 12 hours under nitrogen atmosphere.After the removal of the solvent under reduced pressure, water (300 mL)was added to the residue, and then extracted with ethyl acetate (300mL×3). The combined organic phases were dried over anhydrous sodiumsulfate and concentrated to give the title compound (65 g, yield: 100%).

Step B:

Chloride ammonium (28 g, 525 mmol, 2.5 eq.) and iron powder (58.8 g,1.05 mol, 5.0 eq.) was added to a solution of1-bromo-2-fluoro-4-(3-nitrophenoxy)benzene (65 g, 210 mmol, 1.0 eq.) inethanol (300 mL) and water (60 mL). The reaction solution was refluxedfor 12 hours under nitrogen. After cooling to room temperature, themixture was filtered through Celite. The filtrate concentrated to givethe crude product, which was purified by HPLC (C18 reverse phase column,mobile phase: acetonitrile/water/0.7% NH₄HCO₃, eluent gradient 10%-100%(volume ratio)). After the removal of volatile solvent, the desiredfraction was lyophilized to give the title compound (19 g, yield: 23%).

Step C:

3-(4-bromo-3-fluorophenoxy)benzenamine (9 g, 32 mmol, 1.0 eq.) was addedto pyridine-hydrogen fluoride solution (30 mL) in portions at −10° C.The resulting reaction mixture was stirred at 0° C. for 30 minutes,cooled to −10° C., and then sodium nitrite (2.42 g, 35 mmol, 1.1 eq.)was added portion-wise. The reaction was stirred at 20° C. for 30minutes, then at 60° C. for 14 hours. After cooling to room temperature,the mixture was poured into ice-ethanol (50 mL), diluted with saturatedsolution of NaHCO₃ (50 mL), and then extracted with ethyl acetate (50mL×3). The combined organic phases were dried over anhydrous sodiumsulfate and concentrated to give the crude product, which was purifiedby silica gel column chromatography(eluent:petroleum ether) to give thetitle compound (5.8 g, yield: 64%).

Step D:

1-bromo-2-fluoro-4-(3-fluorophenoxy) benzene (5.8 g, 20 mmol, 1.0 eq.),bis pinacolato boronate (6.1 g, 24 mmol, 1.2 eq.), potassium acetate(3.9 g, 40 mmol, 2.0 eq.) and [1,1′-bis (diphenylphosphino) ferrocene]dichloropalladium (0.89 g, 1.2 mmol, 0.06 eq.) were dissolved in1,4-dioxane (100 mL). The reaction mixture was stirred at 85° C. for 14hours under nitrogen atmosphere. After cooling to room temperature, themixture was filtered through Celite. The filtrate was concentrated togive the crude product, which was purified by silica gel columnchromatography (eluent: petroleum ether) to give the title compound (6.5g, yield: 100%).

Step E:

(R)-tert-butyl3-(4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyrrolidine-1-carboxylate(6.5 g, 15.0 mmol, 1.0 eq.),2-(2-fluoro-4-(3-fluorophenoxy)phenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane(6.5 g, 19.6 mmol, 1.3 eq.), potassium phosphate (6.4 g, 30.1 mmol, 2.0eq.) and Pd-118 (0.25 g, 0.39 mmol, 0.01 eq.) were added to a mixture of1,4-dioxane/water (16 mL, 1/1, v/v). The resulting mixture was stirredat 85° C. for 12 hours under nitrogen atmosphere. After cooling to roomtemperature, the reaction mixture was diluted with water (50 mL), andthen extracted with ethyl acetate (100 mL×3). The combined organicphases were dried over anhydrous sodium sulfate and concentrated to givethe crude product, which was purified by silica gel columnchromatography (eluent: ethyl acetate) to give the title compound (4.2g, yield: 55%).

Step F:

HCl/EA (10 mL, 4 mol/L) was added to a solution of (3R)-tert-butyl3-(4-amino-3-(2-fluoro-4-(3-fluorophenoxy)phenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyrrolidine-1-carboxylate(4.2 g, 8.27 mmol) in dichloromethane (15 mL) at 0° C. The reaction wasstirred for 1 hour at room temperature, and then concentrated underreduced pressure to give the title compound hydrochloride (3.7 g, yield:92%).

Step G:

Sodium hydroxide (10%, 15.3 mL) and acryloyl chloride (0.67 g, 7.44mmol, 0.9 eq.) were sequentially added dropwise to a solution of3-(2-fluoro-4-(3-fluorophenoxy)phenyl)-1-((R)-pyrrolidin-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine(3.7 g, 8.27 mmol, 1.0 eq.) in tetrahydrofuran (20 mL) at 0° C. Thereaction was stirred at room temperature for 10 minutes, quenched withsaturated NaHCO₃ (20 mL), and extracted with dichloromethane (30 mL×3).The combined organic phases were dried over anhydrous sodium sulfate andconcentrated to give the crude product, which was purified by silica gelcolumn chromatography (eluent: petroleum ether:ethyl acetate=1:0 to 1:1)to give the title compound (2.5 g, yield: 65%).

LC/MS (method: UFLC): RT=3.178 min; m/z=463.0 [M+H]⁺; Total runningtime=7.000 min.

¹H NMR (400 MHz, CDCl₃) δ 8.36 (s, 1H), 7.53-7.49 (m, 1H), 7.40-7.35 (m,1H), 6.95-6.81 (m, 4H), 6.41-6.39 (m, 2H), 5.69-5.55 (m, 3H), 4.14-3.98(m, 3H), 3.78-3.72 (m, 1H), 2.71-2.54 (m, 2H).

Compound 6

Step A:

A mixture of propiolic acid (1 g, 14.28 mmol, 1.0 eq.) and HBr (40%aqueous solution, 1.7 mL, 0.88 eq.) was stirred overnight at 140° C.Solvent was distilled off under reduced pressure. The obtained crudeproduct was crystallized from water (4 mL×3) to give the title compound(0.76 g, yield: 35%).

¹H NMR (400 MHz, CDCl₃) δ 7.76 (d, J=14 Hz, 1H), 6.55 (d, J=14 Hz, 1H).

Step B:

Na-Hg (6 g, 49.67 mmol, 2.5 eq.) was added to a solution of(E)-3-bromoacrylic acid (3 g, 19.87 mmol, 1.0 eq.) in D₂O (30 mL) at0˜5° C. The reaction was stirred at room temperature for 36 hours. Theaqueous phase was adjusted pH=5 with 1M hydrochloric acid, and thenextracted with diethyl ether (20 mL×5). The combined organic phases weredried over anhydrous sodium sulfate and concentrated under reducedpressure to give the title compound (0.52 g, yield: 36%).

¹H NMR (400 MHz, CDCl₃) δ 7.76 (d, J=17.2 Hz, 1H), 6.55 (d, J=17.2 Hz,1H).

Step C:

(E)-3-deuteriumacrylic acid (76 mg, 1.08 mmol, 1.0 eq.), HATU (530 mg,1.40 mmol, 1.3 eq.) and N, N-diisopropylethylamine (419 mg, 3.24 mmol,3.0 eq.) were added to a solution of3-(2-fluoro-4-(2,3,5,6-tetrafluorophenoxy)phenyl)-1-((R)-pyrrolidin-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine(500 mg, 1.08 mmol, 1.0 eq.) in dichloromethane (50 mL). The reactionwas stirred at room temperature for 12 hours, and concentrated to givethe crude product, which was purified by HPLC-separation (instrument: LC8A & Gilson 215, fraction collector column: Synergi Max-RP 150*30 mm*4u,mobile phase A: water (0.5% HCl), mobile phase B: acetonitrile, flowrate: 30 mL/min, gradient B: 36%˜37%, 0˜17 minutes). After the removalof volatile solvent, the desired fraction was lyophilized to give thetitle compound hydrochloride (76 mg, yield: 13%).

LC/MS (method: UFLC): RT=2.765 min; m/z=518.1 [M+H]⁺; Total runningtime=7.000 min.

¹H NMR (400 MHz, CD₃OD) δ 8.41 (s, 1H), 7.66 (t, J=8.4 Hz, 1H),7.51-7.44 (m, 1H), 7.09-7.01 (m, 2H), 6.66-6.56 (m, 1H), 6.28-6.23 (m,1H), 5.75-5.66 (m, 1H), 4.19-4.16 (m, 1H), 4.06-4.02 (m, 1.5H),3.89-3.85 (m, 1H), 3.78-3.72 (m, 0.5H), 2.63-2.49 (m, 2H).

Compound 7

Step A:

A mixture of propiolic acid (1 g, 14.28 mmol, 1.0 eq.) and HBr (40%aqueous solution, 1.7 mL, 0.88 eq.) was stirred overnight at 55° C.Solvent was distilled off under reduced pressure. The obtained crudeproduct was crystallized from petroleum ether (4 mL×3) to give the titlecompound (0.3 g, yield: 14%).

¹H NMR (400 MHz, CDCl₃) δ 7.16 (d, J=8.4 Hz, 1H), 6.67 (d, J=8.4 Hz,1H).

Step B:

Na-Hg (6 g, 49.67 mmol, 2.5 eq.) was added to a solution of(Z)-3-bromoacrylic acid (3 g, 19.87 mmol, 1.0 eq.) in D₂O (30 mL) at0˜5° C. The reaction was stirred at room temperature for 36 hours. Theaqueous phase was adjusted pH=5 with 1M hydrochloric acid, and thenextracted with diethyl ether (20 mL×5). The combined organic phases weredried over anhydrous sodium sulfate and concentrated under reducedpressure to give the title compound (0.34 g, yield: 23%).

¹H NMR (400 MHz, CDCl₃) δ 6.14 (d, J=10.4 Hz, 1H), 5.96 (d, J=10.4 Hz,1H).

Step C:

(Z)-3-deuteriumacrylic acid (151 mg, 2.16 mmol, 1.0 eq.), HATU (1.06 g,2.80 mmol, 1.3 eq.) and N, N-diisopropylethylamine (838 mg, 6.48 mmol,3.0 eq.) were added to a solution of3-(2-fluoro-4-(2,3,5,6-tetrafluorophenoxy)phenyl)-1-((R)-pyrrolidin-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine(1.0 g, 2.16 mmol, 1.0 eq.) in dichloromethane (50 mL). The reaction wasstirred at room temperature for 12 hours, and concentrated to give thecrude product, which was purified by HPLC-separation (instrument: LC 8A& Gilson 215, fraction collector column: Synergi Max-RP 150*30 mm*4u,mobile phase A: water (0.5% HCl), mobile phase B: acetonitrile, flowrate: 30 mL/min, gradient B: 36%˜37%, 0˜17 minutes). After the removalof volatile solvent, the desired fraction was lyophilized to give thetitle compound hydrochloride (228 mg, yield: 20%).

LC/MS (method: UFLC): RT=2.775 min; m/z=518.1 [M+H]⁺; Total runningtime=7.000 min.

¹H NMR (400 MHz, CD₃OD) δ 8.45 (s, 1H), 7.70 (t, J=8.4 Hz, 1H),7.52-7.46 (m, 1H), 7.13-7.05 (m, 2H), 6.71-6.61 (m, 1H), 5.80-5.73 (m,2H), 4.23-4.20 (m, 1H), 4.09-4.04 (m, 1.5H), 3.93-3.90 (m, 1H),3.80-3.75 (m, 0.5H), 2.67-2.56 (m, 2H).

Compound 20

A mixture of3-[2-fluoro-4-(3-fluorophenoxy)phenyl]-1-[(3R)-pyrrolidin-3-yl]pyrazolo[3,4-d]pyrimidin-4-amine(200.00 mg, 489.72 umol, 1.00 eq.), but-2-ynoic acid (41.17 mg, 489.72umol, 1.00 eq.), HATU (93.10 mg, 244.86 umol, 0.50 eq.) and DIPEA (75.95mg, 587.66 umol, 102.64 uL, 1.20 eq.) in DCM (5.00 mL) was stirred at15-18° C. for 2 hrs. TLC showed starting material consumed. The mixturewas evaporated to dryness. The residue was purified by prep-HPLC(column: Boston Green ODS 150*30 5u; mobile phase:acetonitrile/water/0.05% HCl, gradient: 22%-52% (volume ratio), time: 12min) to give the title compound as hydrochloride salt (82.00 mg, yield:32.77%).

LC/MS (Method: UFLC): RT=3.057 min; m/z=475.0 [M+H]⁺; Total running time7.000 min.

¹H NMR (400 MHz, CDCl₃) δ 9.92 (s, 1H), 8.34 (d, J=8.8 Hz, 1H), 7.56(br, 1H), 7.41-7.36 (m, 1H), 7.00-6.86 (m, 5H), 6.58 (br, 1H), 5.62-5.58(m, 1H), 4.22-3.74 (m, 4H), 2.65-2.50 (m, 2H), 2.02-1.96 (m, 3H).

Example 2 In Vitro Assay

Inhibition Assay of BTK Kinase Activity:

The enzyme reaction mixture of BTK wild type standard HTRF assaycontained 1 nM BTK wild type, 1 μM biotin-TK1 peptide, and 30 μM ATP ina buffer. The enzyme reaction were carried out at room temperature for60 minutes. 5 μl of 0.2 M EDTA were added to quench the reaction andthen the inhibitors (5 μl) were added at final concentrations of 2 nMantibody and 62.5 nM XL665. The plates were incubated at roomtemperature for 60 minutes and then read in the Envision plate reader.The readouts were transformed into inhibition rate % by the equation of(Min Ratio)/(Max-Min)*100%. Hence the IC50 data of test compounds weregenerated by using four parameters curve fitting.

TABLE 2 Assay data for representative compounds Com- BTK Com- BTK Com-BTK pound IC₅₀ pound IC₅₀ pound IC₅₀ No. (μM) No. (μM) No. (μM) 1 0.0022 0.023 3 0.0005 4 0.021 5 0.001Inhibition Assay of Tumor Cell Activity:

Tumor cells (TMD-8, DoHH2 and WSU-DLCL2) were transferred and attachedto 96-well plates. After one night, blank buffer and selectedconcentrations (0.01 nM-100 μM) of the test compound solution wereadded. After 48 hours incubation, CellTiter-Go was added to lyse thecells. Recording luminescent signal and calculate the percent inhibitionof cell viability.

TABLE 3 Inhibition of individual compound on TMD-8 cell line (Inh %)Compound Name Compound (Mechanism) Inh % 100 uM 10 uM 1 uM 0.1 uM 0.01uM 3 3 AVG 99.69 74.93 61.66 59.59 46.07 (BTK) SD 0.10 0.64 3.97 1.491.60 3 3 AVG 55.05 52.40 51.66 (BTK) SD 3.47 2.17 1.21 6 6 AVG 99.3862.52 60.21 52.99 32.29 (BTK) SD 0.09 1.58 3.62 3.53 5.50 7 7 AVG 99.3262.89 58.57 58.68 33.85 (BTK) SD 0.13 2.18 0.90 2.20 3.05 8 IdelalisibAVG 96.79 87.69 68.92 48.83 29.44 (PI3K) SD 0.27 1.07 2.87 1.35 3.83 9Ibrutinib AVG 99.93 81.41 66.11 59.14 56.16 (BTK) SD 0.01 2.27 2.07 1.772.47 10 Ruxolitinib AVG 100.78 −5.53 0.05 (JAK1/2) SD 0.05 10.51 10.0011 Tofactinib AVG 6.66 1.31 6.02 (JAK3) SD 7.59 8.82 14.08 12 ABT-199AVG 37.55 18.81 11.12 Venetoclax SD 3.83 5.60 2.80 (Bcl-2) 13 OTS-964AVG 101.20 49.10 8.71 (TOPK) SD 0.08 4.49 10.40 14 Everolimus AVG 68.5967.64 65.55 (mTOR) SD 1.71 2.76 2.35 15 Pomalidomide AVG 76.84 61.6945.12 (IMID) SD 1.03 2.34 1.26 16 Lenalidomide AVG 94.21 21.04 7.43(IMID) SD 0.46 5.67 2.61 17 Rapamycin AVG 64.99 59.83 58.71 (mTOR) SD2.77 1.45 2.37 18 Methotrexate AVG 39.55 32.04 −8.45 (antifolate) SD0.71 3.05 6.02 19 Ceritinib AVG (ALK) SD Note: AVG: Average; SD:Standard Deviation

Table 4 below demonstrates that double combination has significantinhibition of tumor cell viability. The combination of Compounds 8 and13; the combination of Compounds 14 and 13; the combination of Compounds15 and 13; and the combination of Compounds 3 and 13 have shown thehighest inhibitory activity against TMD-8 cells.

TABLE 4 Inhibition of “two in one” composition on TMD-8 cell line (Inh%) Comp. @Conc. Inh % 3 @1 uM 3 @0.1 uM 3 @0.01 uM 14 @0.1 uM AVG 65.9467.20 66.17 SD 1.41 0.73 1.64 15 @0.1 uM AVG 53.25 49.26 30.27 SD 3.190.67 2.67  8 @0.1 uM AVG 68.05 64.71 63.56 SD 2.04 2.50 5.10 13 @0.1 uMAVG 82.60 68.80 77.27 SD 3.50 2.64 1.91 17 @0.1 uM AVG 75.73 80.41 75.12SD 0.53 1.29 6.22 12 @0.1 uM AVG 85.97 79.99 65.36 SD 1.50 1.54 0.83 18@0.1 uM AVG 59.93 46.58 35.68 SD 2.77 6.76 5.94 Comp. @Conc. Inh % 8 @1uM 8 @0.1 uM 8 @0.01 uM 14 @0.1 uM AVG 81.20 67.95 58.69 SD 0.33 1.591.08 15 @0.1 uM AVG 76.96 42.58 24.14 SD 0.95 7.50 3.94 13 @0.1 uM AVG95.76 83.60 75.38 SD 0.31 0.53 3.51  3 @0.1 uM AVG 86.26 80.21 73.01 SD2.25 2.87 2.46 Comp. @Conc. Inh % 13 @1 uM 13 @0.1 uM 13 @0.01 uM  8@0.1 uM AVG 99.31 47.73 48.71 SD 0.06 2.52 4.50 14 @0.1 uM AVG 99.4659.47 60.30 SD 0.11 0.73 1.44 15 @0.1 uM AVG 99.09 8.82 12.97 SD 0.173.93 4.84  3 @0.1 uM AVG 99.16 97.60 52.43 SD 0.42 0.19 1.07 Comp.@Conc. Inh % 15 @1 uM 15 @0.1 uM 15 @0.01 uM  8 @0.1 uM AVG 80.19 43.3442.81 SD 1.25 5.76 3.81 14 @0.1 uM AVG 61.84 57.04 58.46 SD 1.62 1.700.32 13 @0.1 uM AVG 71.75 30.72 1.36 SD 0.35 7.16 5.17  3 @0.1 uM AVG96.92 70.04 49.93 SD 0.19 4.46 5.42 Comp. @Conc. Inh % 18 @1 uM 18 @0.1uM 18 @0.01 uM 14 @0.1 uM AVG 54.43 52.67 56.87 SD 0.70 2.71 2.27  3@0.1 uM AVG 46.90 42.73 34.99 SD 2.34 2.91 1.26 Comp. @Conc. Inh % 9 @1uM 9 @0.1 uM 9 @0.01 uM 14 @0.1 uM AVG 71.04 58.89 56.54 SD 2.52 9.7113.33 19 @0.1 uM AVG 52.60 43.68 33.70 SD 3.67 4.16 1.51 18 @0.1 uM AVG55.19 42.42 32.13 SD 2.63 3.32 3.08

Table 5 below demonstrates that triple combination has significantinhibition of tumor cell viability. The combination of Compounds 3, 14and 12; and the combination of Compound 3, 8 and 12 have shown thehighest inhibitory activity up to 95% even at the concentration as lowas 10 nM for Compound 3.

TABLE 5 Inhibition of “three in one” composition on TMD-8 cell line (Inh%) Comp. @Conc. Inh % 3 @1 uM 3 @0.1 uM 3 @0.01 uM 14 @0.1 uM + AVG76.42 80.77 83.22 15 @0.1 uM SD 4.50 1.38 0.37 17 @0.1 uM + AVG 89.8985.62 88.57 15 @0.1 uM SD 0.72 7.68 3.37 14 @0.1 uM + AVG 93.44 94.7394.65 12 @0.1 uM SD 0.55 0.92 1.11 8 @0.1 uM + AVG 95.56 95.30 94.62 12@0.1 uM SD 0.40 0.10 0.06 18 @0.1 uM + AVG 66.44 71.70 58.27 14 @0.1 uMSD 8.75 1.91 2.80 Comp. @Conc. Inh % 15 @1 uM 15 @0.1 uM 15 @0.01 uM 14@0.1 uM + AVG 92.74 82.66 75.17 3 @0.1 uM SD 0.38 1.90 2.48 Comp. @Conc.Inh % 8 @1 uM 8 @0.1 uM 8 @0.01 uM 14 @0.1 uM + AVG 87.17 79.06 59.45 15@0.1 uM SD 1.70 0.73 2.16 Comp. @Conc. Inh % 13 @1 uM 13 @0.1 uM 13@0.01 uM 15 @0.1 uM + AVG 98.97 22.27 −19.34 3 @0.1 uM SD 0.28 34.1811.80 15 @0.1 uM + AVG 99.30 29.58 −3.32 8 @0.1 uM SD 0.15 27.38 11.2715 @0.1 uM + AVG 99.33 19.51 −1.30 14 @0.1 uM SD 0.11 48.40 6.76 Comp.@Conc. Inh % 10 @1 uM 10 @0.1 uM 10 @0.01 uM 15 @0.1 uM + AVG 24.40−6.35 −0.77 3 @0.1 uM SD 5.84 5.66 18.61 15 @0.1 uM + AVG 16.26 2.31−6.21 8 @0.1 uM SD 2.14 1.28 4.86 15 @0.1 uM Plus AVG −0.86 0.98 −2.4014 @0.1 uM SD 6.50 5.87 1.06 Comp. @Conc. Inh % 9 @1 uM 9 @0.1 uM 9@0.01 uM 18 @0.1 uM + AVG 72.55 66.59 64.76 14 @0.1 uM SD 0.22 11.128.34 15 @0.1 uM Plus AVG 82.81 86.91 78.60 14 @0.1 uM SD 1.05 1.41 13.08

Table 6 below demonstrates that the triple combination of Compound 3, 14and 15 is effective against multi-drug resistant WSU-DLCL2 tumor cells,superior to each single agent alone.

TABLE 6 Inhibition of individual compound and “three in one” compositionon resistant WSU-DLCL2 cell line (Inh %) Comp. @Conc. Inh % 1 uM 0.1 uM0.01 uM 3 AVG 41.04 −1.36 −10.06 SD 7.73 2.59 11.14 15 AVG 46.61 −8.32−14.53 SD 1.15 4.49 13.72 14 AVG 55.35 46.71 40.81 SD 1.67 0.53 2.67Comp. @Conc. Inh % 3 3 3 15 @0.1 uM + AVG 83.76 63.69 56.67 14 @0.1 uMSD 1.33 4.19 4.36

Table 7 below demonstrates that the triple combination of Compounds 3,14 and 15 is effective against more difficult to treat DoHH-2 tumorcells, superior to each of the single agents alone.

TABLE 7 Inhibition of individual compound and “three in one” compositionon DoHH-2 cell line (Inh %) Comp. @Conc. Inh % 1 uM 0.1 uM 0.01 uM 3 AVG49.93 34.83 15.58 SD 2.72 0.70 5.54 15 AVG 51.32 6.75 −7.95 SD 3.86 8.771.57 14 AVG 60.33 59.17 51.80 SD 3.52 1.68 3.35 Comp. @Conc. Inh % 3 3 315 @0.1 uM + AVG 78.75 81.87 71.87 14 @0.1 uM SD 0.45 1.02 6.47

Table 8 demonstrates that the triple combination of Compound 3, 14 and15 at various doses of each single agent are all effective against thesensitive TMD-8 tumor cells.

TABLE 8 Inhibition of compositions with different proportions on TMD-8cell line (Inh %) Molar ratio Comp. @Conc. Inh % 1.0 uM 0.1 uM 0.01 uM3 + 14 AVG 72.97 71.71 66.64   (19:1 molar ratio) SD 0.93 1.49 0.83 3 +14 + 15 AVG 97.08 89.29 67.75 (19:1:37 molar ratio)  SD 0.52 1.30 1.123 + 14 + 15 AVG 97.16 91.23 80.09  (1:1:1 molar ratio) SD 0.17 0.85 0.963 + 14 + 15 AVG 78.21 72.73 63.34 (50:1:1 molar ratio) SD 2.26 1.21 0.973 + 14 + 15 AVG 88.09 77.18 68.76 (10:1:1 molar ratio) SD 0.70 1.66 2.123 @1 uM 3 @0.1 uM 3 @0.01 uM 14 @0.1 uM + AVG 85.33 88.83 87.78 15 @0.1uM SD 0.78 0.71 2.36

Example 3 In Vivo Assay

Pharmacokinetic Study in Male SD Rats:

Male SD rats for pharmacokinetic study within 24 hours were divided intotwo groups: intravenous administration and oral administration. Eachgroup has three animals. For group of intravenous administration, bloodsamples were collected at pre-dose, 0.0833, 0.167, 0.5, 1, 2, 4, 8, 24 hpost-dose; for group of oral administration, blood samples werecollected at pre-dose, 0.167, 0.5, 1, 2, 4, 8, 24 h post-dose. Afterblood collection, HPLC-MS/MS was applied to determine plasmaconcentrations of the compound. The calculated pharmacokineticparameters of intravenous group include mean plasma clearance (CLp),mean apparent volume of distribution at stead state (Vdss), 0-24 h areaunder the curve (AUC), 0-24 h mean residence time (MRT), the half-life(T1/2); The calculated pharmacokinetic parameters of oral group includemean peak concentration (Cmax), 0-24 h area under the curve (AUC), 0-24h mean residence time (MRT); mean relative bioavailability for thestudy.

Pharmacokinetic Study in Beagle Dogs: Beagle dogs for pharmacokineticstudy within 24 hours were divided into two groups: intravenousadministration (1 mg per kilogram) and oral administration (3 mg perkilogram). Each group has three animals. For group of intravenousadministration, blood samples were collected at pre-dose, 0.033, 0.083,0.25, 0.5, 1, 3, 6, 9, 24 h post-dose; for group of oral administration,blood samples were collected at pre-dose, 0.083, 0.25, 0.5, 1, 3, 6, 9,24 h post-dose. After blood collection, HPLC-MS/MS was applied todetermine plasma concentrations of the compound. The calculatedpharmacokinetic parameters of intravenous group include mean plasmaclearance (CLp), mean apparent volume of distribution at stead state(Vdss), 0-24 h area under the curve (AUC), 0-24 h mean residence time(MRT), the half-life (T1/2); The calculated pharmacokinetic parametersof oral group include mean peak concentration (Cmax), 0-24 h area underthe curve (AUC), 0-24 h mean residence time (MRT); mean relativebioavailability for the study.

TABLE 9 PK Parameters for Compound 3 in rats Group 1 2 Dose Route IV PODose level 2 mg/kg 10 mg/kg Mean SD Mean SD C₀ or C_(max) (ng/mL) 1390247 641 191 T_(max) (hr) — — 1.33 0.753 T_(1/2) (hr) 0.787 0.0895 1.710.489 Vdss (L/kg) 1.61 0.339 — — CL (mL/min/kg) 20.2 5.60 — —AUC_(0-last) (hr · ng/mL) 1740 421 3230 1120 AUC_(0-inf) (hr · ng/mL)1740 420 3260 1140 Bioavailability (%)^(a) — — 37.1 —

TABLE 10 PK Parameters for Compound 3 in dogs Group 1 2 Dose Route IV PODose level 2 mg/kg 5 mg/kg Mean SD Mean SD C₀ or C_(max) (ng/mL) 66379.5 189 53.3 T_(max) (hr) — — 1.17 0.408 T_(1/2) (hr) 2.27 0.873 2.921.22 Vdss (L/kg) 4.24 0.370 — — CL (mL/min/kg) 34.6 5.58 — —AUC_(0-last) (hr · ng/mL) 977 181 650 247 AUC_(0-inf) (hr · ng/mL) 987183 574 123 Bioavailability (%)^(a) — — 26.2 —

Table 11 below shows that the AUC of Compound 3 in rats is significantlyhigher than that of ibrutinib (U.S. FDA's NDA Application No.205552Orig1s000_pharmacological review(s)).

TABLE 11 TK data for Compound 3 in rats Dose Study Cmax TmaxAUC_(0-24 h) (mg/kg) Day Sex (ng/mL) (h) (h*ng/mL) 40 1 Male 2160 2.013700 Female 2660 1.0 17300 28 Male 2090 2.0 15400 Female 2970 1.0 17300100 1 Male 2740 2.0 21700 Female 3700 4.0 28900 28 Male 3990 2.0 30300Female 3830 1.0 29600 200 1 Male 4220 2.0 37600 Female 4680 4.0 65200 28Male 4540 2.0 45100 Female 5490 8.0 60200

Table 12 below shows that the AUC of Compound 3 in dogs is significantlyhigher than that of ibrutinib (U.S. FDA's NDA Application No.205552Orig1s000_pharmacological review(s)).

TABLE 12 TK data for Compound 3 in dogs Dose (mg/kg/ Study Cmax TmaxAUC_(0-24 h) day) Day Sex (ng/mL) (h) (h*ng/mL) 15 1 Male  746 ± 18.12.0 (1.0-2.0) 3550 ± 562 Female  685 ± 212 1.0 (1.0-2.0) 2930 ± 980 28Male  576 ± 145 2.0 (2.0-2.0) 3260 ± 732 Female  687 ± 123 2.0 (1.0-2.0)3730 ± 549 45 1 Male 1240 ± 381 2.0 (1.0-2.0)  6480 ± 1670 Female 1220 ±431 2.0 (2.0-2.0)  6220 ± 3000 28 Male 1470 ± 538 2.0 (2.0-4.0)  9170 ±3810 Female 1060 ± 263 2.0 (2.0-4.0)  8130 ± 1490 105 28 Male 2700 ± 7692.0 (2.0-2.0) 16400 ± 5410 Female 2420 ± 670 2.0 (2.0-4.0) 17300 ± 2830150 1 Male 2460 ± 858 4.0 (1.0-8.0)  22900 ± 13900 Female 1850 ± 605 2.0(1.0-4.0) 11200 ± 5990Inhibition Study of Tumor Growth In Vivo:

SCID mice or nude mice (weighing about 18 g at the beginning of theexperiment) were randomly divided into groups by the software in orderto achieve close average weights between groups and control the biaswithin the allowable range. The mice were injected BTK cell lines(TMD-8, WSU-DLCL2 and DoHH-2) for tumor formation. Inhibitors wereadministered orally once or twice a day, a total of 14 days, 21 days or28 days. Body weights and tumor volume were recorded.

Xenograft Tumor Models Inoculated with TMD-8, or DoHH2, or WSU-DLCL2Tumor Cell Lines:

TMD-8 is a sensitive human diffuse large B-cell lymphoma cell line, andDoHH2 is a more difficult to treat human follicular lymphoma cell line,while WSU-DLCL2 is a multi-drug resistant (MDR) human non-Hodgkin'slymphoma cell line. Drug combination therapies provide better efficaciesin all three tumor models than single targeted agent alone.

Compounds (Compounds 3, 9, 14 and others as shown in the charts) and itscombinations were evaluated against tumor growth in xenograft models infemale CB-17 SCID mice. The TMD-8, DoHH2, WSU-DLCL2 tumor cells weremaintained in vitro as a suspension culture in RPMI-1640 mediumsupplemented with 10% heat inactivated fetal calf serum at 37° C. in anatmosphere of 5% CO₂ in air. The tumor cells were routinely subculturedtwice weekly. The cells growing in an exponential growth phase wereharvested and counted for tumor inoculation. Each mouse was inoculatedsubcutaneously at the right flank with the tumor cells (10×10⁶) in 0.2ml of PBS with Matrigel (1:1) for tumor development. The treatments werestarted after the average tumor size reached approximately 100-200 mm³.Each group consisted of 6-10 tumor-bearing mice. The testing article(vehicle, compound or combination) was orally administrated to the miceaccording to the predetermined doses for 14-days or 21-days. Animal bodyweight and tumor volume were measured every 2- or 3-days throughout thetreatment.

Adjuvant-Induced Arthritis AA Model:

The combination of Compounds 3 and 14 was evaluated in adjuvant-inducedarthritis (AA) model in female Lewis rats. All rats except the normalgroup were immunized with complete Freund's adjuvant (CFA)subcutaneously at the left hind paw to induce arthritis at day 0. At 6days post immunization, some rats started to display clinical symptomsof arthritis, e.g., erythema and swelling. At day 13, the immunizedanimals were re-grouped to 7 groups, including vehicle, Compound 3 (5mg/kg)/Compound 14 (0.5 mg/kg) BID treatment, Compound 3 (15mg/kg)/Compound 14 (1.5 mg/kg) BID treatment, Compound 3 (30mg/kg)/Compound 14 (3 mg/kg) QD treatment, Compound 3 (5 mg/kg) BIDtreatment, Compound 14 (0.5 mg/kg) BID treatment, and a positive control(Compound 11, 3 mg/kg, BID treatment) groups, based on body weight andclinical scores. The treatments were given orally for 3 consecutiveweeks. The body weight, paw volume and clinical score were monitoredevery other day after day 13 throughout the course of the study. At theterminal point, right hind paws were collected for histopathologyanalysis with H.E. staining.

Collagen-Induced Arthritis (CIA) Model:

The combination of Compounds 3 and 12 was evaluated in collagen inducedarthritis (CIA) mouse model in Male DBA/1 mice. The animals were dividedinto 8 groups, including a normal, a vehicle, five treatment groups: Allanimals (except the normal group) were immunized with 200 μg of bovinecollagen (type II) on day 0 and day 21. Seven days (day 28) afterboosting immunization, animals started to show symptoms of disease withan average clinical score around 1. On the same day, immunized mice wererandomly divided into 7 groups: Compound 3 (1.5 mg/kg) and Compound 14(0.15 mg/kg) combination treatment BID group, Compound 3 (4.5 mg/kg) andCompound 14 (0.45 mg/kg) combination treatment BID group, Compound 3(1.5 mg/kg) and Compound 14 (0.15 mg/kg) combination treatment QD group,Compound 3 (1.5 mg/kg) single treatment QD group, Compound 14 (0.15mg/kg) single treatment QD group, and a positive control group (0.2mg/kg of dexamethasone), and start to dosing and treatment. Thetreatments were given orally for 2 consecutive weeks. Body weight andclinical score were monitored through the study (recorded three times aweek started after second immunization). At the end of the study,animals were euthanized and both hind paws were collected forhistopathology analysis.

As shown in FIG. 4, although the administration of an mTOR kinaseinhibitor (e.g., Compound 14) led to tumor disappearance in TMD-8diffuse large B-cell lymphoma (DLBCL) mice model after 15 days oftreatment, the tumor rebounded after treatment was stopped after day 15.Surprisingly, no tumor rebound was observed when the mTOR kinaseinhibitor was administered in combination with a BTK inhibitor (e.g.,Compound 3). In comparison, as shown in FIGS. 2-4, the DLBCL tumor didnot disappear when the combination of the BTK inhibitor and an IMiD(e.g., Compounds 15 and 16) was administered, when the combination ofthe BTK inhibitor and a PI3K kinase inhibitor (e.g., Compound 8) wasadministered, when the combination of the IMiD and the PI3K kinaseinhibitor was administered, or when the combination of the BTKinhibitor, the IMiD and the PI3K kinase inhibitor was administered.

As shown in FIG. 5, the triple combination of a BTK inhibitor (e.g.,Compound 3), an mTOR kinase inhibitor (e.g., Compound 14), and an IMiD(e.g., Compound 15) led to tumor disappearance in TMD-8 mice model after9 days of treatment and, unexpectedly, the tumor did not rebound aftertreatment was stopped after day 12 and completed regression was observedthroughout the rest of the 21-day period.

As shown in FIG. 12, the triple combination of a BTK inhibitor (e.g.,Compound 3), an mTOR kinase inhibitor (e.g., Compound 14), and a Bcl-2inhibitor (e.g., Compound 12) led to tumor disappearance in TMD-8 micemodel after 8 days of treatment and, unexpectedly, the tumor did notrebound after treatment was stopped and completed regression wasobserved throughout the rest of the 14-day period. In comparison, theDLBCL tumor did not disappear when only the mTOR kinase inhibitor andthe Bcl-2 inhibitor were administered in combination, when only the BTKinhibitor and the Bcl-2 inhibitor were administered in combination, orwhen the BTK inhibitor and the Bcl-2 inhibitor were administered incombination with a PI3K kinase inhibitor (e.g., Compound 8).

As shown in FIGS. 6 and 7, the administration of a BTK inhibitor (e.g.,Compound 3) and an mTOR kinase inhibitor (e.g., Compound 14) performedbetter than the administration of the BTK inhibitor alone in terms ofcontrolling tumor growth in DoHH2 follicular lymphoma (FL) mice model.Surprisingly, when the BTK inhibitor and the mTOR kinase inhibitor wereadministered in combination with an IMiD (e.g., Compound 15), tumorgrowth in DoHH2 mice model was reduced to a minimal level. Suchsynergistic effects were reproduced in two separate experiments (FIGS. 6and 7).

As shown in FIG. 8, the efficacy of Compound 3 in WSU-DLCL2non-Hodgkin's lymphoma mice model is in line with the efficacy ofIbrutinib, an FDA-approved BTK inhibitor. As shown in FIG. 9, thecombination of a BTK inhibitor (e.g., Compound 3), an mTOR kinaseinhibitor (e.g., Compound 14), and an IMiD (e.g., Compound 15) at atotal of 21 mg/kg/day for all three drugs combined were able to reducetumor growth in WSU-DLCL2 mice model. In comparison, EZH2 inhibitorEPZ-6438, which is currently undergoing clinical trial, achieved asimilar effect against the WSU-DLCL tumor only by gavage to mice at avery high dose of 480 mg/kg/day (see Knutson et al., Mol. Cancer Ther.,2014, 13:842-854, FIG. 4A).

Moreover, FIG. 10 demonstrates that the efficacy of the triplecombination therapy against TMD-8 diffuse large B-cell lymphoma remainssubstantially consistent at various doses of the individual ingredients.Furthermore, FIG. 11 demonstrates that the efficacy of the triplecombination therapy against DoHH2 follicular lymphoma is not limited toCompound 3 but applicable to other BTK inhibitors (e.g., Ibrutinib) aswell.

As shown in FIG. 13, the combination of a BTK inhibitor (e.g., Compound3) and an mTOR kinase inhibitor (e.g., Compound 14) at various dosessynergistically reduced the paw volume of AA rats model, exhibitingefficacies similar to the positive control (Tofactinib). Such treatmenteffects are absent from rats treated with Compound 3 alone or Compound14 alone.

As shown in FIG. 15, the combination of a BTK inhibitor (e.g., Compound3) and an mTOR kinase inhibitor (e.g., Compound 14) at various dosessynergistically reduced the clinical score of CIA mice model, exhibitingefficacies similar to the positive control (Dexamethasone). Suchtreatment effects are absent from mice treated with Compound 3 alone orCompound 14 alone.

Additional data of the BTK Compounds are shown in the tables below.Table 13 below demonstrates that low dose combination is synergisticagainst tumor cells and has shown synthetic lethality. At much lowerdose than each single agent, the triple combination of Compounds 3, 14,and 15 resulted in completed tumor regression in 9 days while the doublecombination of Compounds 3 and 14 required 15 days. Single agent wasmuch less effective or causing tumor to rebound when the administrationof such agent is stopped. No tumor rebound was seen with the triplecombination after tumor regression even when the administration of thetriple combination is switched to the vehicle.

TABLE 13 Antitumor effect of Single-drug and combination therapyCorresponding Compound Antitumor effect FIG. Dose (mg/kg) (%) FIG. 1Oral, Vehicle control — BID, 1 (10 mg/kg) 56 14 days 1 (30 mg/kg) 77 3(10 mg/kg) 64 3 (30 mg/kg) 82 3 (90 mg/kg) 93 FIG. 2 Oral, Vehiclecontrol — BID, 3 (10 mg/kg) 63 14 days 3 (30 mg/kg) 89 15 (30 mg/kg) 243 (10 mg/kg) 95 15 (30 mg/kg) FIG. 3 Oral, Vehicle control — BID, 3 (5mg/kg) 90 21 days 3 (10 mg/kg) 96 15 (10 mg/kg) −8 8 (10 mg/kg) 26 3 (5mg/kg) 82 15 (10 mg/kg) 3 (10 mg/kg) 89 15 (10 mg/kg) 3 (5 mg/kg) 95 8(10 mg/kg) 3 (10 mg/kg) 98 8 (10 mg/kg) 3 (5 mg/kg) 94 15 (10 mg/kg) 8(5 mg/kg) 3 (10 mg/kg) 94 15 (10 mg/kg) 8 (10 mg/kg) 15 (10 mg/kg) 86 8(10 mg/kg) FIG. 4 Oral, Vehicle control — BID, 3 (10 mg/kg) 77 21 days16 (10 mg/kg) 16 16 (30 mg/kg) 42 14 (1 mg/kg) 97 (Tumor rebounded atday 17.) 14 (3 mg/kg) 99 (Tumor rebounded at day 19.) 3 (10 mg/kg) 100(Tumor 14 (1 mg/kg) didn't rebound from day 15.) 3 (10 mg/kg) 100 (Tumor14 (3 mg/kg) didn't rebound from day 15.) 3 (10 mg/kg) 72 16 (10 mg/kg)3 (10 mg/kg) 80 16 (30 mg/kg) FIG. 5 Oral, Vehicle control — BID, 3 (5mg/kg) 33 21 days 3 (5 mg/kg) 100 (Tumor 15 (5 mg/kg) disappeared 14(0.5 mg/kg) completely at day 9 and didn't rebound.) FIG. 10 Oral,Vehicle control — BID, 3 (5 mg/kg) 100 (Tumor 14 days 15 (5 mg/kg)disappeared 14 (0.5 mg/kg) completely at day 10 and didn't rebound.) 3(10 mg/kg) 100 (Tumor 15 (1 mg/kg) disappeared 14 (0.5 mg/kg) completelyat day 10 and didn't rebound.) 3 (20 mg/kg) 100 (Tumor 15 (1 mg/kg)disappeared 14 (0.5 mg/kg) completely at day 10 and didn't rebound.)FIG. 7 Oral, Vehicle control — BID, 3 (5 mg/kg)  15.9 21 days 3 (5mg/kg)  80.3 15 (5 mg/kg) 14 (0.5 mg/kg) FIG. 6 Oral, Vehicle control —BID, 3 (5 mg/kg)  28.8 21 days 3 (10 mg/kg)  20.1 3 (30 mg/kg)  35.6 3(5 mg/kg)  58.3 14 (0.5 mg/kg) 3 (5 mg/kg)  79.4 15 (5 mg/kg) 14 (0.5mg/kg) FIG. 8 Oral, Vehicle control — BID, 9 (30 mg/kg) 24 28 days 3 (10mg/kg) 22 3 (30 mg/kg) 30 3 (45 mg/kg) 32 FIG. 9 Oral, Vehicle control —BID, 3 (5 mg/kg)  4 18 days 3 (10 mg/kg)  7 3 (30 mg/kg) 23 3 (5 mg/kg)44 14 (0.5 mg/kg) 3 (5 mg/kg) 50 15 (5 mg/kg) 14 (0.5 mg/kg) FIG. 11Oral, Vehicle control — BID, 3 (5 mg/kg) 63 14 days 15 (5 mg/kg) 14 (0.5mg/kg) 9 (4.3 mg/kg) 67 15 (5 mg/kg) 14 (0.5 mg/kg) FIG. 12 Oral,Vehicle control — BID, 3 (5 mg/kg) 75 14 days 12 (5 mg/kg) 12 8 (10mg/kg) 48 3 (5 mg/kg) 86 12 (5 mg/kg) 8 (10 mg/kg) 37 12 (5 mg/kg) 12 (5mg/kg) 77 14 (0.5 mg/kg) 3 (5 mg/kg) 100  12 (5 mg/kg) 14 (0.5 mg/kg) 3(5 mg/kg) 89 12 (5 mg/kg) 8 (10 mg/kg)

Table 14 demonstrates that the lose dose combination is safe without anysignificant body weight changes between all treated and control groups.

TABLE 14 Animal weight for 3/14/15 and 9/14/15 combination therapy DayWeight (g) 0 2 5 7 9 12 14 Vehicle control Mean 22.9 22.4 22.8 23.0 23.723.5 23.6 SEM 0.5 0.5 0.6 0.5 0.6 0.5 0.6  3 (5 mg/kg) Mean 21.9 21.622.8 22.5 22.6 22.5 22.3 15 (5 mg/kg) SEM 0.4 0.4 0.3 0.4 0.5 0.5 0.6 14(0.5 mg/kg)  9 (4.3 mg/kg) Mean 22.2 21.7 22.7 22.9 23.1 22.7 22.6 15 (5mg/kg) SEM 0.5 0.5 0.6 0.5 0.4 0.4 0.6 14 (0.5 mg/kg)

Table 14 demonstrates that the lose dose combination at various doses ofeach agent is safe without any significant body weight changes betweenall treated and control groups.

TABLE 15 Animal weight for 3/14/15 combination therapy Day Weight (g) 02 5 7 9 12 14 Vehicle control Mean 21.2 21.1 21.1 21.4 21.5 21.8 21.9SEM 0.5 0.4 0.4 0.5 0.4 0.5 0.5  3 (5 mg/kg) Mean 22.1 21.8 21.8 21.821.7 22.3 21.7 15 (5 mg/kg) SEM 0.8 0.7 0.8 0.7 0.8 0.8 0.7 14 (0.5mg/kg)  3 (10 mg/kg) Mean 21.6 21.5 21.8 22.2 22.4 22.4 22.1 15 (1mg/kg) SEM 0.5 0.6 0.6 0.7 0.7 0.6 0.7 14 (0.5 mg/kg)  3 (20 mg/kg) Mean21.4 21.0 21.1 21.3 21.4 21.3 21.4 15 (1 mg/kg) SEM 0.6 0.5 0.5 0.6 0.60.5 0.6 14 (0.5 mg/kg)

Table 16 below shows that not all triple combinations have superiorsynergistic effects, further evidencing that the synergistic effectswith the triple combinations of BTK/mTOR/IMiD and BTK/mTOR/Bcl-2 areunexpected. Both in vitro and in vivo synergistic effects suppressingcancer cells have been achieved with these two triple combinations.

TABLE 16 Lack of inhibition on tumor cell viability for triplecombination of JAK1 inhibition with BTK/IMid, IMiD/PI3K and IMid/mTORComp. @Conc. Inh % 10 @1 uM 10 @0.01 uM 10 @0.01 uM 15 @0.1 uM + AVG24.40 −6.35 −0.77 3 @0.1 uM SD 5.84 5.66 18.61 15 @0.1 uM + AVG 16.262.31 −6.21 8 @0.1 uM SD 2.14 1.28 4.86 15 @0.1 uM + AVG −0.86 0.98 −2.414 @0.1 uM SD 6.5 5.87 1.06

Tables 17-20 below show that double combination is more effective thansingle agent alone in autoimmune animal models.

TABLE 17 Paw volume of the animals during the AA study Day Paw volume(mL) 0 17 26 28 31 33 Normal Mean 1.00 1.08 1.05 1.05 1.05 1.04 SEM 0.030.02 0.01 0.02 0.02 0.02 Vehicle control Mean 1.12 2.18 2.71 2.71 2.692.70 SEM 0.09 0.09 0.15 0.11 0.11 0.11  3 (5 mg/kg) Mean 1.03 1.77*1.65*** 1.69*** 1.63*** 1.53*** 14 (0.5 mg/kg) SEM 0.02 0.07 0.08 0.080.08 0.07  3 (15 mg/kg) Mean 1.01 1.62*** 1.45*** 1.44*** 1.34***1.29*** 14 (1.5 mg/kg) SEM 0.02 0.10 0.11 0.10 0.09 0.08  3 (30 mg/kg)Mean 1.01 1.63*** 1.50*** 1.45*** 1.41*** 1.38*** 14 (3 mg/kg) SEM 0.010.08 0.09 0.08 0.08 0.07  3 (5 mg/kg) Mean 1.04 1.87 ns 2.29** 2.12***2.12*** 2.09*** SEM 0.01 0.12 0.14 0.19 0.19 0.20 14 (0.5 mg/kg) Mean1.02 1.84 ns 2.01*** 2.04*** 1.94*** 1.93*** SEM 0.01 0.08 0.13 0.120.11 0.10 11 (3 mg/kg) Mean 1.00 1.54*** 1.37*** 1.33*** 1.23*** 1.23***SEM 0.02 0.08 0.08 0.07 0.05 0.05 *p < 0.05, **p < 0.01, ***p < 0.001

TABLE 18 Pathological score of the animals during the AA studyPathological score (Mean ± SEM) Inflammatory Pannus Cartilage Bone Groupcell infiltration growth injury resorption Total Normal 0.0 ± 0.00 0.0 ±0.00 0.0 ± 0.00 0.0 ± 0.00  0.0 ± 0.00 Vehicle control  4 ± 0.00  4 ±0.00 3.8 ± 0.13 3.7 ± 0.15 15.5 ± 0.27 3 (5 mg/kg) 3.5 ± 0.22 2.8 ± 0.252.6 ± 0.27 2.8 ± 0.29 11.7 ± 0.96 14 (0.5 mg/kg) 3 (15 mg/kg) 2.9 ± 0.311.6 ± 0.22 1.1 ± 0.28 2.2 ± 0.49   7.8 ± 1.21*** 14 (1.5 mg/kg) 3 (30mg/kg) 2.6 ± 0.37 2.2 ± 0.44 1.7 ± 0.40 2.4 ± 0.40   8.9 ± 1.55*** 14 (3mg/kg) 3 (5 mg/kg) 3.5 ± 0.34 3.2 ± 0.53 2.8 ± 0.51 3.0 ± 0.45 12.5 ±1.78 14 (0.5 mg/kg) 3.9 ± 0.10 3.7 ± 0.21 3.5 ± 0.27 3.5 ± 0.17 14.6 ±0.54 11 (3 mg/kg) 1.9 ± 0.18 0.4 ± 0.22 0.2 ± 0.20 0.5 ± 0.22   3.0 ±0.73*** ***p < 0.001, v.s. Vehicle, Kruskal-Wallis test, Dunn's post-hoctest

TABLE 19 Clinical Score after twenty-first days after the firstimmunization during the CIA Study Clinical Score day 21 32 39 42 NormalMean 0.00 0.00 0.00 0.00 SEM 0.00 0.00 0.00 0.00 Vehicle control Mean0.00 4.00 7.6 8.00 SEM 0.00 0.86 1.14 1.20 Dexamethasone (0.2 mg/kg)Mean 0.00 0.60*** 0.40*** 0.20*** SEM 0.00 0.34 0.22 0.20 3 (1.5 mg/kg)Mean 0.00 1.60* 1.40*** 1.60*** 14 (0.15 mg/kg) BID SEM 0.00 0.45 0.370.40 3 (4.5 mg/kg) Mean 0.00 0.40*** 0.2*** 0.10*** 14 (0.45 mg/kg) BIDSEM 0.00 0.16 0.13 0.10 3 (1.5 mg/kg) Mean 0.00 1.50** 1.40*** 1.60***14 (0.15 mg/kg) QD SEM 0.00 0.40 0.50 0.58 3 (1.5 mg/kg) QD Mean 0.002.60 4.20*** 4.00*** SEM 0.00 0.86 1.14 1.22 14 (0.15 mg/kg) QD Mean0.00 4.00 5.60 5.70* SEM 0.00 0.54 0.82 0.80 *p < 0.05, **p < 0.01, ***p< 0.001, v.s. Vehicle, Two-way ANOVA, Bonferrni's post-hoc test

TABLE 20 Pathological score of the animals during the CIA studyPathological score (Mean ± SEM) Inflammatory cell Pannus Cartilage BoneGroup infiltration growth injury resorption Total Vehicle control Lefthind 1.60 ± 0.65 1.30 ± 0.56 1.40 ± 0.58 1.00 ± 0.42 15.50 ± 2.30 pawRight hind 2.80 ± 0.61 2.40 ± 0.54 2.50 ± 0.56 2.50 ± 0.56 pawDexamethasone Left hind 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00*** (0.2 mg/kg) paw QD Right hind 0.00 ± 0.00 0.00 ± 0.000.00 ± 0.00 0.00 ± 0.00 paw  3 (1.5 mg/kg) Left hind 0.50 ± 0.22 0.20 ±0.20 0.20 ± 0.20 0.10 ± 0.10  1.50 ± 0.78*** 14 (0.15 mg/kg) paw BIDRight hind 0.20 ± 0.20 0.10 ± 0.10 0.10 ± 0.10 0.10 ± 0.10 paw  3 (4.5mg/kg) Left hind 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00  0.00 ±0.00*** 14 (0.45 mg/kg) paw BID Right hind 0.00 ± 0.00 0.00 ± 0.00 0.00± 0.00 0.00 ± 0.00 paw  3 (1.5 mg/kg) Left hind 0.20 ± 0.20 0.20 ± 0.200.20 ± 0.20 0.20 ± 0.20  0.80 ± 0.80*** 14 (0.15 mg/kg) paw QD Righthind 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 paw  3 (1.5 mg/kg)Left hind 1.00 ± 0.47 0.80 ± 0.47 0.80 ± 0.47 0.60 ± 0.13  3.90 ±1.80*** QD paw Right hind 0.20 ± 0.20 0.20 ± 0.20 0.20 ± 0.20 0.10 ±0.10 paw 14 (0.15 mg/kg) Left hind 2.10 ± 0.64 2.00 ± 0.67 2.00 ± 0.671.80 ± 0.61 15.90 ± 4.50 QD paw Right hind 2.00 ± 0.67 2.00 ± 0.67 2.00± 0.67 2.00 ± 0.67 paw Normal Left hind 0.00 ± 0.00 0.00 ± 0.00 0.00 ±0.00 0.00 ± 0.00  0.00 ± 0.00 paw Right hind 0.00 ± 0.00 0.00 ± 0.000.00 ± 0.00  0.00 ± 0.00 paw ***p < 0.001, v.s. Vehicle, Kruskal-Wallistest, Dunn's post-hoc test

Table 21 below shows the structures of some of the compounds useful inthe present invention.

TABLE 21 Compound Entry Code Name Structure  1 ACP-196 Acalabrutinib

 2 AVL-292 (CC-292)

 3 ONO-4059

 4 HM71224 Olmutinib

 5 RN486

 6 CNX-774

 7 XL388

 8 GDC-0349

 9 AZD2014 Vistusertib

10 AZD8055

11 MLN0128 Sapanisertib

12 CC-122

13 CC-220

14 PF-05212384 Gedatolisib

15 GDC-0980 Apitolisib

16 GSK2126458

17 BEZ235

18 IPI-145 Duvelisib

19 CAL-101 Idelalisib

20 ABT-199 Venetoclax

21 BI-97C1 sabutoclax

22 OTS964

23 CH5424802 Alectinib

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Exemplary methods and materialsare described below, although methods and materials similar orequivalent to those described herein can also be used in the practice ortesting of the present invention. All publications and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. Although a number of documents are cited herein, this citationdoes not constitute an admission that any of these documents forms partof the common general knowledge in the art.

Throughout this specification and embodiments, the word “comprise,” orvariations such as “comprises” or “comprising” will be understood toimply the inclusion of a stated integer or group of integers but not theexclusion of any other integer or group of integers. The materials,methods, and examples are illustrative only and not intended to belimiting.

As used herein, the terms “substantially,” “substantial,” and “about”are used to describe and account for small variations. When used inconjunction with an event or circumstance, the terms can refer toinstances in which the event or circumstance occurs precisely as well asinstances in which the event or circumstance occurs to a closeapproximation. For example, the terms can refer to less than or equal to±10%, such as less than or equal to ±5%, less than or equal to ±4%, lessthan or equal to ±3%, less than or equal to ±2%, less than or equal to±1%, less than or equal to ±0.5%, less than or equal to ±0.1%, or lessthan or equal to ±0.05%.

Additionally, amounts, ratios, and other numerical values are sometimespresented herein in a range format. It is to be understood that suchrange format is used for convenience and brevity and should beunderstood flexibly to include numerical values explicitly specified aslimits of a range, but also to include all individual numerical valuesor sub-ranges encompassed within that range as if each numerical valueand sub-range is explicitly specified. For example, a ratio in the rangeof about 1 to about 200 should be understood to include the explicitlyrecited limits of about 1 and about 200, but also to include individualratios such as about 2, about 3, and about 4, and sub-ranges such asabout 10 to about 50, about 20 to about 100, and so forth.

What is claimed is:
 1. A method for treating a lymphoma, comprisingadministering to a subject in need thereof a therapeutically effectiveamount of (a) a Bruton tyrosine kinase (BTK) inhibitor, (b) a mammaliantarget of rapamycin (mTOR) kinase inhibitor, and (c) a B-cell lymphoma 2(Bcl-2) inhibitor.
 2. The method of claim 1, wherein the BTK inhibitoris a compound represented by Formula I, II, Ia, Ib, IIa, or IIb,

wherein: each R¹ is F; R² is F; R³ is H; n is 1, 2, 3 or 4; and m is 1or 2, or an enantiomer, a diastereomer, a pharmaceutically acceptablesalt, or a prodrug thereof.
 3. The method of claim 1, wherein the BTKinhibitor is selected from the group consisting of Compound 1, Compound2, Compound 3, Compound 4, and Compound 5, as shown in Table 1, or anenantiomer, a diastereomer, a pharmaceutically acceptable salt, or aprodrug thereof.
 4. The method of claim 1, wherein the BTK inhibitor isibrutinib or a pharmaceutically acceptable salt thereof.
 5. The methodof claim 1, wherein the mTOR kinase inhibitor is everolimus or apharmaceutically acceptable salt thereof.
 6. The method of claim 1,wherein the mTOR kinase inhibitor is rapamycin or a pharmaceuticallyacceptable salt thereof.
 7. The method of claim 1, wherein the Bcl-2inhibitor is venetoclax or a pharmaceutically acceptable salt thereof.8. The method of claim 1, wherein the lymphoma is selected from thegroup consisting of diffuse large B-cell lymphoma, marginal zonelymphoma, chronic lymphocytic leukemia (CLL), Waldenström'sMacroglobulinemia (WM), and mantle cell lymphoma (MCL).
 9. The method ofclaim 4, wherein the Bcl-2 inhibitor is venetoclax.
 10. The method ofclaim 9, wherein the mTOR kinase inhibitor is everolimus.
 11. The methodof claim 9, wherein the mTOR kinase inhibitor is rapamycin or apharmaceutically acceptable salt thereof.
 12. The method of claim 4,wherein the mTOR kinase inhibitor is everolimus.
 13. The method of claim12, wherein the Bcl-2 inhibitor is navitoclax.
 14. The method of claim7, wherein the BTK inhibitor is acalabrutinib, and the mTOR kinaseinhibitor is everolimus or a pharmaceutically acceptable salt thereof.15. The method of claim 9, wherein the mTOR kinase inhibitor is AZD2014or a pharmaceutically acceptable salt thereof.
 16. The method of claim7, wherein the BTK inhibitor is acalabrutinib, and the mTOR kinaseinhibitor is rapamycin or a pharmaceutically acceptable salt thereof.17. The method of claim 7, wherein the BTK inhibitor is acalabrutinib,and the mTOR kinase inhibitor is AZD2014 or a pharmaceuticallyacceptable salt thereof.